Fused aminodihydrothiazine derivatives

ABSTRACT

The present invention relates to a fused aminodihydrothiazine derivative of formula (I): 
                         
wherein
         X is hydrogen or fluorine;   A is CH or N;   Y is methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl, methoxy, ethoxy, methoxymethyl or —C≡N;
 
and pharmaceutically acceptable salts thereof;
 
which compound has an Aβ production inhibitory effect or a BACE1 inhibitory effect and is useful as a prophylactic or therapeutic agent for a neurodegenerative disease caused by Aβ and typified by Alzheimer-type dementia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/354,716, filed on Jan. 20, 2012, which issued as U.S. Pat. No.8,338,407 on Dec. 25, 2012, which claims the benefit of priority ofBritish application serial number 1101140.0, filed Jan. 21, 2011. Thedisclosure of the prior application is considered part of (and isincorporated by reference in) the disclosure of this application.

The present invention relates to a fused aminodihydrothiazine derivativeand pharmaceutical use thereof. More particularly, the present inventionrelates to a fused aminodihydrothiazine derivative which has anamyloid-β (hereinafter referred to as Aβ protein production inhibitoryeffect or a beta-site amyloid-β precursor protein cleavage enzyme 1(hereinafter referred to as BACE1 or beta-secretase) inhibitory effectand is effective for treating a neurodegenerative disease caused by Aβprotein, in particular, Alzheimer-type dementia, Down's syndrome or thelike, and to a pharmaceutical composition comprising the fusedaminodihydrothiazine derivative as an active ingredient.

Alzheimer's disease is a disease characterized by degeneration and lossof neurons as well as formation of senile plaques and neurofibrillarytangles. Currently, only the symptoms of Alzheimer's disease are treatedusing a symptom-improving agent typified by an acetylcholinesteraseinhibitor, and a fundamental remedy to inhibit progression of thedisease has not yet been developed. It is necessary to develop a methodfor controlling the cause of the onset of pathology in order to create afundamental remedy for Alzheimer's disease.

It is assumed that Aβ-proteins as breakdown products of amyloidprecursor proteins (hereinafter referred to as APP) are highly involvedin degeneration and loss of neurons and onset of symptoms of dementia.Aβ-proteins have, as main components, Aβ40 consisting of 40 amino acidsand Aβ42 with two amino acids added at the C-terminal. The Aβ40 and Aβ42are known to be highly prone to aggregation and to be the maincomponents of senile plaques. Further, it is known that the Aβ40 andAβ42 are increased by mutations in APP and presenilin genes which isobserved in familial Alzheimer's disease. Accordingly, a compound thatreduces production of Aβ40 and Aβ42 is expected to be a diseaseprogression inhibitor or prophylactic agent for Alzheimer's disease.

Aβ is produced by the cleavage of APP by beta-secretase (BACE1) andsubsequently by gamma-secretase. For this reason, attempts have beenmade to create gamma-secretase and beta-secretase inhibitors in order toinhibit Aβ production.

Published International patent application WO2011/005738 (Eli Lilly andCompany) describes compounds of formula (A) and their use as BACEinhibitors:

where R¹, R², R³, X, m, n and p are defined therein.

Fused aminodihydrothiazine compounds of formula (B) have already beendisclosed in published International patent application WO2009/091016(Eisai R&D Management Co., Ltd.):

wherein ring A represents a C₆₋₁₄aryl group or the like; L represents—NR^(e)CO— [wherein R^(e) represents a hydrogen atom or the like] or thelike; ring B represents a C₆₋₁₄aryl group or the like; X represents aC₁₋₃alkylene group or the like; Y represents a single bond or the like;Z represents a C₁₋₃alkylene group or the like; R¹ and R² independentlyrepresent a hydrogen atom or the like; and R³, R⁴, R⁵ and R⁶independently represent a hydrogen atom, a halogen atom or the like.

Further fused aminodihydrothiazine compounds of formula (C) have beendisclosed in published International patent application WO2010/038686(Eisai R&D Management Co., Ltd.):

wherein ring A represents a C₆₋₁₄aryl group or the like; L represents—NR^(e)CO—[wherein R^(e) represents a hydrogen atom or the like] or thelike; the ring B represents a C₆₋₁₄aryl group or the like; X representsa C₁₋₃alkylene group or the like; Y represents a single bond or thelike; Z represents an oxygen atom or the like; R¹ and R² eachindependently represents a hydrogen atom or the like; and R³, R⁴, R⁵ andR⁶ each independently represents a hydrogen atom, a halogen atom or thelike.

The present invention represents a selection from the genus of compoundsdisclosed in WO2009/091016.

An object of the present invention is to provide further compounds thathave an Aβ production inhibitory effect or a BACE1 inhibitory effect andare useful as prophylactic or therapeutic agents for a neurodegenerativedisease caused by Aβ and typified by Alzheimer-type dementia, whichcompounds are fused aminodihydrothiazine derivatives.

Thus, the present invention provides the compound of formula (I):

wherein

X is hydrogen or fluorine;

A is CH or N;

Y is methyl, ethyl, monofluoromethyl, difluoromethyl, trifluoromethyl,difluoroethyl, methoxy, ethoxy, methoxymethyl or —C≡N;

and pharmaceutically acceptable salts thereof.

In one embodiment of the present invention, X is hydrogen.

In another embodiment of the present invention, A is N.

In another embodiment of the present invention, Y is methyl,monofluoromethyl, difluoromethyl, trifluoromethyl or methoxy.

One favoured group of compounds of the present invention is the compoundof formula (Ia) and pharmaceutically acceptable salts thereof:

where Y is as hereinbefore defined. Preferably, Y is methyl,monofluoromethyl, difluoromethyl, trifluoromethyl, difluoroethyl,methoxy, ethoxy or methoxymethyl.

In one embodiment the present invention provides a compound of formula(Ia) wherein Y is methoxy or monofluoromethyl.

Another favoured group of compounds of the present invention is thecompound of formula (Ib) and pharmaceutically acceptable salts thereof:

where Y is as hereinbefore defined. Preferably, Y is methyl,monofluoromethyl, difluoromethyl or methoxy.

A further favoured group of compounds of the present invention is thecompound of formula (Ic) and pharmaceutically acceptable salts thereof:

where Y is as hereinbefore defined. Preferably, Y is methyl, ethyl,trifluoromethyl, methoxy or —C≡N.

Preferred compounds of the present invention are:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-cyanopicolinamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(trifluoromethyl)picolinamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methylpyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methylpicolinamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethylpicolinamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypicolinamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethoxypyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(1,1-difluoroethyl)pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(trifluoromethyl)pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(methoxymethyl)pyrazine-2-carboxamide:

-   N-{3-[(4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5-dihydro-4H-furo[3,4    d][1,3]thiazin-7a(7H)-yl]-4-fluorophenyl}-5-[(²H₃)methyloxy]pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-methoxypyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-methylpyrazine-2-carboxamide:

-   N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-(fluoromethyl)-pyrazine-2-carboxamide:

and pharmaceutically acceptable salts thereof.

In one embodiment, the present invention provides a compound which isN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a compound whichisN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof.

Specific compounds within the scope of this invention include thosenamed in the Examples below and their pharmaceutically acceptable salts.

As used herein, the term “difluoroethyl” refers to an alkyl group havingtwo carbon atoms and substituted with two fluorine atoms. Examples ofthe group are CH₃—CF₂—, CH₂F—CHF— and CHF₂—CH₂—. In the presentinvention, the group is preferably CH₃—CF₂—.

The compound of formula (I) is not limited to a specific isomer andincludes all possible isomers (such as a keto-enol isomer, animine-enamine isomer and a rotamer) and mixtures thereof. For example,the compound of formula (I) includes the following tautomers:

The compounds of the present invention contain three chiral centerslocated on the tetrahydrofuro-thiazinyl ring within formula (I). Thestereochemical configuration at each of these chiral centers ispreferably S, i.e. they are (4aS,5S,7aS) stereoisomers. For theavoidance of doubt the (4aS,5S,7aS) stereoisomers of the presentinvention may be present as a mixture with one or more of the otherpossible stereoisomers, for example in a racemic mixture.

In one embodiment, the present invention provides a compound of formula(I) which is stereochemically pure at the (4aS,5S,7aS) chiral centers.In the context of the present specification, the term stereochemicallypure denotes a compound which has 80% or greater by weight of the(4aS,5S,7aS) stereoisomer and 20% or less by weight of otherstereoisomers. In a further embodiment, the compound of formula (I) has90% or greater by weight of the (4aS,5S,7aS) stereoisomer and 10% orless by weight of other stereoisomers. In a yet further embodiment, thecompound of formula (I) has 95% or greater by weight of the (4aS,5S,7aS)stereoisomer and 5% or less by weight of other stereoisomers. In a stillfurther embodiment, the compound of formula (I) has 97% or greater byweight of the (4aS,5S,7aS) stereoisomer and 3% or less by weight ofother stereoisomers.

In the present specification, although crystal polymorphs of thecompound may be present, the compound is similarly not limited theretoand may be present as a single crystal form or a mixture of singlecrystal forms. The compound may be an anhydride or a hydrate. Any ofthese forms is included in the claims of the present specification.

The present invention also includes isotopically-labelled compounds,which are identical to the compounds of formula (I), except that one ormore atoms are replaced by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in nature.Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,fluorine, phosphorous, chlorine, technetium and iodine, such as ²H, ³H,¹¹C, ¹⁴C, ¹³N, ¹⁵O, ¹⁸F, ³²P, ^(99m)Tc, ¹²³I and ¹³¹I.

Compounds of the present invention and pharmaceutically acceptablederivatives (e.g. salts) of said compounds that contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of the present invention. Isotopically-labelled compounds ofthe present invention, for example those into which radioactive isotopessuch as ³H and/or ¹⁴C are incorporated, are useful in drug and/orsubstrate tissue distribution assays. ³H and ¹⁴C are considered usefuldue to their ease of preparation and detectability. ¹¹C, ¹⁵O and ¹⁸Fisotopes are considered useful in PET (positron emission tomography),and ^(99m)Tc, ¹²³I and ¹³¹I isotopes are considered useful in SPECT(single photon emission computerized tomography), all useful in brainimaging. Substitution with heavier isotopes such as ²H can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, are considered useful in some circumstances.Isotopically labelled compounds of formula (I) of this invention cangenerally be prepared by carrying out the procedures disclosed in theSchemes and/or in the Examples below, by substituting a readilyavailable isotopically labelled reagent for a non-isotopically labelledreagent.

The fused aminodihydrothiazine derivative of the formula (I) accordingto the present invention may be a pharmaceutically acceptable salt.Pharmaceutically acceptable salts include those described by Berge,Bighley and Monkhouse, J. Pharm. Sci., 1977, 766, 1-19. Specificexamples of the pharmaceutically acceptable salt include inorganic acidsalts (such as sulfates, nitrates, perchlorates, phosphates, carbonates,bicarbonates, hydrofluorides, hydrochlorides, hydrobromides andhydroiodides), organic carboxylates (such as acetates, oxalates,maleates, tartrates, fumarates, citrates, malonates and lactates),organic sulfonates (such as methanesulfonates,trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates,toluenesulfonates and camphorsulfonates), amino acid salts (such asaspartates and glutamates), quaternary amine salts, alkali metal salts(such as sodium salts and potassium salts) and alkali earth metal salts(such as magnesium salts and calcium salts).

The compound of the formula (I) according to the present invention canbe converted to a pharmaceutically acceptable salt by a conventionalmethod where necessary. The salt can be prepared by a method in whichmethods typically used in the field of organic synthetic chemistry andthe like are appropriately combined. Specific examples of the methodinclude neutralization titration of a free solution of the compound ofthe present invention with an acid solution.

The fused aminodihydrothiazine derivative of the formula (I) orpharmaceutically acceptable salt according to the present invention maybe a solvate thereof. Examples of a solvate include a hydrate.

The compound of the formula (I) according to the present invention canbe converted to a solvate by subjecting the compound to a solvateforming reaction known per se where necessary.

The present invention further provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use in therapy.

The fused aminodihydrothiazine derivative or pharmaceutically acceptablesalt thereof or solvate thereof according to the present invention hasan excellent Aβ production inhibitory effect or BACE1 inhibitory effectand is useful as a prophylactic or therapeutic agent for aneurodegenerative disease caused by Aβ and typified by Alzheimer-typedementia. The compounds of the invention reduce both Aβ40 and Aβ42.Furthermore, the compounds of the present invention may have a BACE 2inhibitory effect.

Thus, in another aspect, the present invention provides a compound offormula (I) as defined above, or a pharmaceutically acceptable saltthereof, for inhibiting production of amyloid-β protein.

In a further aspect, the present invention provides a compound offormula (I) as defined above, or a pharmaceutically acceptable saltthereof, for inhibiting beta-site amyloid-β precursor protein cleavingenzyme 1 (BACE 1).

In a further aspect, the present invention provides a compound offormula (I) as defined above, or a pharmaceutically acceptable saltthereof, for treating a neurodegenerative disease. Examples ofneurodegenerative diseases include Alzheimer-type dementia (AD), Down'ssyndrome, cerebrovascular amyloid angiopathy (CAA), mild cognitiveimpairment (MCI), memory loss, presenile dementia, senile dementia,hereditary cerebral hemorrhage with amyloidosis, and other degenerativedementias such as dementias of mixed vascular and degenerative origin,dementia associated with supranuclear palsy, dementia associated withcortical basal degeneration, dementia associated with Parkinson'sDisease (PD), and dementia associated with diffuse Lewy Body type of AD.In one embodiment, the neurodegenerative disease is Alzheimer-typedementia (AD).

In another aspect, the invention provides the use of a compound offormula (I) as defined above, or a pharmaceutically acceptable saltthereof, for the manufacture of a medicament for the treatment orprevention of a neurodegenerative disease, such as Alzheimer-typedementia (AD), Down's syndrome, cerebrovascular amyloid angiopathy(CAA), mild cognitive impairment (MCI), memory loss, presenile dementia,senile dementia, hereditary cerebral hemorrhage with amyloidosis, andother degenerative dementias such as dementias of mixed vascular anddegenerative origin, dementia associated with supranuclear palsy,dementia associated with cortical basal degeneration, dementiaassociated with Parkinson's Disease (PD), and dementia associated withdiffuse Lewy Body type of AD. In one embodiment, the neurodegenerativedisease is Alzheimer-type dementia (AD).

In another aspect, the invention provides a method of inhibitingproduction of amyloid-β protein and/or of treating or preventing aneurodegenerative disease, such as Alzheimer-type dementia (AD), Down'ssyndrome, cerebrovascular amyloid angiopathy (CAA), mild cognitiveimpairment (MCI), memory loss, presenile dementia, senile dementia,hereditary cerebral hemorrhage with amyloidosis, and other degenerativedementias such as dementias of mixed vascular and degenerative origin,dementia associated with supranuclear palsy, dementia associated withcortical basal degeneration, dementia associated with Parkinson'sDisease (PD), and dementia associated with diffuse Lewy Body type of AD,involving administering to a human subject in need thereof atherapeutically or prophylactically effective amount of a compound offormula (I) or a pharmaceutically acceptable salt thereof. Examples ofneurodegenerative diseases include those listed above. In oneembodiment, the neurodegenerative disease is Alzheimer-type dementia(AD). “Effective amount” means an amount sufficient to cause a benefitto the subject or at least to cause a change in the subject's condition.

Additional conditions which may be treated by the compounds of thepresent invention include type 2 diabetes, Creutzfield-Jakob Disease(CJD), peripheral nerve injury, peripheral neuropathy, progressivesupra-nuclear palsy, stroke, amyotrophic lateral sclerosis (ALS),autoimmune diseases, inflammation, arterial thrombosis, anxietydisorders, psychotic disorders, epilepsy, seizures, convulsions, stressdisorders, vascular amyloidosis, pain, Gerstmann-Straeussler-Scheinkersyndrome, scrapie, encephalopathy, spino cerebellar ataxia, Wilson'sDisease, Graves Disease, Huntington's Disease, Whipple's Disease,Kostmann Disease, glaucoma, hereditary cerebral hemorrhage withamyloidosis, cerebral hemorrhage with amyloidosis, vascular amyloidosis,brain inflammation, fragile X syndrome, stroke, Tourette's syndrome,inclusion body myositis, stress disorders, depression, bipolar disorderand obsessive compulsive disorder.

In one aspect the present invention further provides a compound offormula (I) as defined above, or a pharmaceutically acceptable saltthereof, for treating type 2 diabetes. In a further aspect the presentinvention further provides the use of a compound of formula (I) asdefined above, or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament for the treatment or prevention of type 2diabetes.

In a yet further aspect the present invention further provides a methodof inhibiting production of amyloid-β protein and/or of treating orpreventing type 2 diabetes involving administering to a human subject inneed thereof a therapeutically or prophylactically effective amount of acompound of formula (I) or a pharmaceutically acceptable salt thereof.

A further aspect of the invention provides a pharmaceutical compositioncomprising a compound of formula (I) as defined above, or apharmaceutically acceptable salt thereof, as active ingredient inassociation with a pharmaceutically acceptable carrier. The compositionmay be in any suitable form, depending on the intended method ofadministration. It may for example be in the form of a tablet, capsuleor liquid for oral administration, or of a solution or suspension foradministration parenterally.

The fused aminodihydrothiazine derivative or pharmaceutically acceptablesalt thereof according to the present invention can be formulated by aconventional method. Preferable examples of the dosage form includetablets, coated tablets such as film tablets and sugar-coated tablets,fine granules, granules, powders, capsules, syrups, troches, inhalants,suppositories, injections, ointments, eye drops, nasal drops, ear drops,cataplasms and lotions.

These solid preparations such as tablets, capsules, granules and powderscan contain generally 0.01 to 100 wt %, and preferably 0.1 to 100 wt %of the fused aminodihydrothiazine derivative or pharmaceuticallyacceptable salt thereof according to the present invention as an activeingredient.

The active ingredient is formulated by blending ingredients generallyused as materials for a pharmaceutical preparation and adding anexcipient, a disintegrant, a binder, a lubricant, a colorant and acorrective typically used, and adding a stabilizer, an emulsifier, anabsorbefacient, a surfactant, a pH adjuster, a preservative and anantioxidant where necessary, for example, using a conventional method.Examples of such ingredients include animal and vegetable oils such assoybean oil, beef tallow and synthetic glyceride; hydrocarbons such asliquid paraffin, squalane and solid paraffin; ester oils such asoctyldodecyl myristate and isopropyl myristate; higher alcohols such ascetostearyl alcohol and behenyl alcohol; a silicone resin; silicone oil;surfactants such as polyoxyethylene fatty acid ester, sorbitan fattyacid ester, glycerol fatty acid ester, polyoxyethylene sorbitan fattyacid ester, polyoxyethylene hydrogenated castor oil and apolyoxyethylene-polyoxypropylene block copolymer; water-soluble polymerssuch as hydroxyethylcellulose, polyacrylic acid, a carboxyvinyl polymer,polyethylene glycol, polyvinylpyrrolidone and methylcellulose; loweralcohols such as ethanol and isopropanol; polyhydric alcohols such asglycerol, propylene glycol, dipropylene glycol and sorbitol; sugars suchas glucose and sucrose; inorganic powders such as silicic anhydride,magnesium aluminum silicate and aluminum silicate; and purified water.Examples of the excipient used include lactose, corn starch, saccharose,glucose, mannitol, sorbitol, crystalline cellulose and silicon dioxide.Examples of the binder used include polyvinyl alcohol, polyvinyl ether,methylcellulose, ethylcellulose, gum arabic, tragacanth, gelatin,shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose,polyvinylpyrrolidone, a polypropylene glycol-polyoxyethylene blockcopolymer and meglumine. Examples of the disintegrant used includestarch, agar, gelatin powder, crystalline cellulose, calcium carbonate,sodium bicarbonate, calcium citrate, dextrin, pectin andcarboxymethylcellulose calcium. Examples of the lubricant used includemagnesium stearate, talc, polyethylene glycol, silica and hydrogenatedvegetable oil. Examples of the colorant used include those permitted tobe added to pharmaceuticals. Examples of the corrective used includecocoa powder, menthol, empasm, mentha oil, borneol and cinnamon powder.Obviously, the ingredients are not limited to the above additiveingredients.

For example, an oral preparation is prepared by adding the fusedaminodihydrothiazine derivative or pharmaceutically acceptable saltthereof according to the present invention as an active ingredient, anexcipient and, where necessary, a binder, a disintegrant, a lubricant, acolorant, a corrective and the like, and then forming the mixture intopowder, fine granules, granules, tablets, coated tablets, capsules orthe like by a conventional method. Obviously, tablets or granules may beappropriately coated, for example, sugar coated, where necessary.

For example, a syrup or an injection preparation is prepared by adding apH adjuster, a solubilizer, an isotonizing agent and the like, and asolubilizing agent, a stabilizer and the like where necessary by aconventional method. The injection may be a previously preparedsolution, or may be powder itself or powder containing a suitableadditive, which is dissolved before use. The injection can containusually 0.01 to 100 wt %, and preferably 0.1 to 100 wt % of the activeingredient. Further, a liquid preparation for oral administration suchas a suspension or a syrup can contain usually 0.01 to 100 wt %, andpreferably 0.1 to 100 wt % of the active ingredient.

For example, an external preparation can be prepared by any conventionalmethod without specific limitations. As a base material, any of variousmaterials usually used for a pharmaceutical, a quasi drug, a cosmetic orthe like can be used. Examples of the base material include materialssuch as animal and vegetable oils, mineral oils, ester oils, waxes,higher alcohols, fatty acids, silicone oils, surfactants, phospholipids,alcohols, polyhydric alcohols, water-soluble polymers, clay minerals andpurified water. A pH adjuster, an antioxidant, a chelator, apreservative and fungicide, a colorant, a flavor or the like can beadded where necessary. Further, ingredients such as an ingredient havinga differentiation inducing effect, a blood flow enhancer, a bactericide,an antiphlogistic, a cell activator, vitamin, amino acid, a humectantand a keratolytic agent can be blended where necessary.

The dose of the fused aminodihydrothiazine derivative orpharmaceutically acceptable salt thereof according to the presentinvention varies according to the degree of symptoms, age, sex, bodyweight, mode of administration, type of salt and specific type ofdisease, for example. Typically, the active ingredient is orallyadministered to an adult at about 30 μg to 10 g, preferably 100 μg to 5g, and more preferably 100 μg to 1 g per day, or is administered to anadult by injection at about 30 μg to 1 g, preferably 100 μg to 500 mg,and more preferably 100 μg to 300 mg per day, in one or several doses,respectively.

Compounds of the formula (I) may be used in combination with othertherapeutic agents, for example medicaments claimed to be useful aseither disease modifying or symptomatic treatments of aneurodegenerative disease such as Alzheimer's disease. Thus, in afurther aspect, the present invention provides a pharmaceutical productcomprising, in combination, a first active ingredient which is acompound of formula (I) or a pharmaceutically acceptable salt thereofand at least one further active ingredient useful in treating aneurodegenerative disease. In one embodiment of the invention, theneurodegenerative disease is Alzheimer-type dementia (AD). Suitableexamples of such further active ingredients may be symptomatic agents,for example those known to modify cholinergic transmission such as M1and M3 muscarinic receptor agonists or allosteric modulators, M2muscarinic antagonists, M4 agonists or positive allosteric modulators(PAMs), acetylcholinesterase inhibitors (such astetrahydroaminoacridine, donepezil hydrochloride and rivastigmine),nicotinic receptor agonists or allosteric modulators (such as α7agonists or allosteric modulators or α4β2 agonists or allostericmodulators), PPAR agonists (such as PPARγ agonists), 5-HT₄ receptoragonists or partial agonists, histamine H3 antagonists, 5-HT₆ receptorantagonists or 5HT₇ receptor ligands and NMDA receptor antagonists ormodulators, 5-HT_(2A) antagonists, 5-HT₇ antagonists, D1 agonists orPAMs, D4 agonists or PAMs, D5 agonists or PAMs, GABA-A α5 inverseagonists or negative allosteric modulators (NAMs), GABA-A α2/3 agonistsor PAMs, mGluR2 modulators (PAMs or NAMs), mGluR3PAMs, mGluR5PAMs, PDE 1inhibitors, PDE 2 inhibitors, PDE 4 inhibitors, PDE 5 inhibitors, PDE 9inhibitors, PDE 10 inhibitors, GlyT1 inhibitors, DAAO inhibitors, ASC1inhibitors, AMPA modulators, SIRT1 activators or inhibitors, AT4antagonists, GalR1 antagonists, GalR3 ligands, adenosine A1 antagonists,adenosine A2a antagonists, α2A antagonists or agonists, selective andunselective norepinephrine reuptake inhibitors (SNRIs), or potentialdisease modifying agents such as gamma secretase inhibitors ormodulators, alpha secretase activators or modulators, amyloidaggregation inhibitors, amyloid antibodies, tau aggregation inhibitorsor tau phosphorylation/kinase inhibitors, taudephosphorylation/phosphatase activators, mitogen-activated proteinkinase kinase 4 (MKK4/MEK4/MAP2K4) inhibitors, c-Jun N-terminal kinase(INK) inhibitors, casein kinase inhibitors, MK2 (mitogen activatedprotein kinase-activated protein kinase 2) inhibitors, MARK (microtubuleaffinity regulating kinase) inhibitors, CDKS (cyclin dependent kinase 5)inhibitors, GSK-3 (glycogen synthase kinase-3) inhibitors andtau-tubulin kinase-1 (TTBK1) inhibitors. Further examples of such othertherapeutic agents may be calcium channel blockers, HMG-CoA(3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitors (statins) andlipid lowering agents, NGF (nerve growth factor) mimics, antioxidants,GPR3 ligands, plasmin activators, neprilysin (NEP) activators, IDE(insulin degrading enzyme) activators, melatonin MT1 and/or MT2agonists, TLX/NR2E1 (tailless X receptor) ligands, GluR1 ligands, RAGE(receptor for advanced glycation end-products) antagonists, EGFR(epidermal growth factor receptor) inhibitors, FPRL-1 (formylpeptide-like receptor-1) ligands, GABA antagonists, and MICAL (moleculeinteracting with casL) inhibitors, e.g. oxoreductase inhibitors, CB1antagonists/inverse agonists, non-steroidal anti-inflammatory drugs(NSAIDs), anti-inflammatory agents (for example agents that could beused to treat neuroinflammation either by enhancing or reducingneuroinflammation), amyloid precursor protein (APP) ligands,anti-amyloid vaccines and/or antibodies, agents that promote or enhanceamyloid efflux and/or clearance, histone deacetylase (HDAC) inhibitors,EP2 antagonists, 11-beta HSD1 (hydroxysteroid dehydrogenase) inhibitors,liver X receptor (LXR) agonists or PAMs, lipoprotein receptor-relatedprotein (LRP) mimics and/or ligands and/or enhancers and/or inhibitors,butyryl cholinesterase inhibitors, kynurinic acid antagonists and/orinhibitors of kynurenine aminotransferease (KAT), orphanin FQ/nociceptin(NOP)/opioid-like receptor 1 (ORL1) antagonists, excitatory amino acidtransporter (EAAT) ligands (activators or inhibitors), and plasminogenactivator inhibitor-1 (PAI-1) inhibitors, niacin and/or GPR109 agonistsor PAMs in combination with cholesterol lowering agents and/or HMGCoAreductase inhibitors (statins), dimebolin or similar agents,antihistamines, metal binding/chelating agents, antibiotics, growthhormone secretagogues, cholesterol lowering agents, vitamin E,cholesterol absorption inhibitors, cholesterol efflux promoters and/oractivators, and insulin upregulating agents.

In one embodiment, the present invention provides a pharmaceuticalproduct comprising, in combination, a first active ingredient which is acompound of formula (I) or a pharmaceutically acceptable salt thereofand at least one further active ingredient selected from:—

-   -   cholinesterase inhibitors, e.g. donepezil, galantamine,        rivastigamine, tetrahydroaminoacridine and pharmaceutically        acceptable salts thereof,    -   5-HT₆ antagonists, e.g. SB-742457 and pharmaceutically        acceptable salts thereof,    -   HMGCoA reductase inhibitors e.g. lovastatin, rosuvastatin,        atorvastatin, simvastatin, fluvastatin, pitavastatin,        pravastatin and pharmaceutically acceptable salts thereof.

The individual components of such combinations may be administeredeither sequentially or simultaneously in separate or combinedpharmaceutical formulations. Consequently, the pharmaceutical productmay, for example be a pharmaceutical composition comprising the firstand further active ingredients in admixture. Alternatively, thepharmaceutical product may for example comprise the first and furtheractive ingredients in separate pharmaceutical preparations suitable forsimultaneous, sequential or separate administration to a patient in needthereof.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier or excipient comprise a furtheraspect of the invention.

When a compound of formula (I) or a pharmaceutically acceptable saltthereof is used in combination with a second therapeutic agent active,the dose of each compound may differ from that when the compound is usedalone. Appropriate doses will be readily appreciated by those skilled inthe art.

Thus, an additional aspect of the invention provides a method ofpreparation of a pharmaceutical composition, involving admixing at leastone compound of formula (I) as defined above, or a pharmaceuticallyacceptable salt thereof, with one or more pharmaceutically acceptableadjuvants, diluents or carriers and/or with one or more othertherapeutically or prophylactically active agents.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising a compound of formula (I) or a pharmaceuticallyacceptable salt thereof, one or more other agents for the treatment ofAlzheimer's disease, such as an M1 and M3 muscarinic receptor agonist orallosteric modulator, an M2 muscarinic antagonist, anacetylcholinesterase inhibitor, a nicotinic receptor agonist orallosteric modulator, a PPAR agonist, a 5-HT4 receptor agonist orpartial agonist, a histamine H3 antagonist, a 5-HT₆ receptor antagonist,a 5HT_(1A) receptor ligand, a NMDA receptor antagonist or modulator, a5-HT_(2A) antagonist, a 5-HT₇ antagonist, a D1 agonist or positiveallosteric modulator (PAM), a D4 agonist or PAM, a GABA-A a5 inverseagonist or negative allosteric modulator (NAM), a GABA-A a2/3 agonist orPAM, a mGluR2 modulator (PAM or NAM), a mGluR3 PAM, a mGluR5 PAM, a PDE1 inhibitor, a PDE 2 inhibitor, a PDE 4 inhibitor, a PDE 5 inhibitor, aPDE 9 inhibitor, a PDE 10 inhibitor, a G1yT1 inhibitor, a DAAOinhibitor, a ASC1 inhibitor, a AMPA modulator, a SIRT1 activator orinhibitor, a AT4 antagonist, a GalR1 antagonist, a GalR3 ligand, anadenosine A1 antagonist, an adenosine A2a antagonist, an α2A antagonistor agonist, a selective or unselective norepinephrine reuptake inhibitor(SNRT), a gamma secretase inhibitor or modulator, an alpha secretaseactivator or modulator, an amyloid aggregation inhibitor, an amyloidantibody, a tau aggregation inhibitor, a tau phosphorylation inhibitor,a MK2 (mitogen activated protein kinase-activated protein kinase 2)inhibitor, a MARK (microtubule affinity regulating kinase) inhibitor, aCDKS (cyclin dependent kinase 5) inhibitor, a GSK-3 (glycogen synthasekinase-3) inhibitor, a calcium channel blocker, a HMG-CoA(3-hydroxy-3-methyl-glutaryl-CoA) reductase inhibitor (statin) and alipid lowering agent, a NGF (nerve growth factor) mimic, an antioxidant,a GPR3 ligand, a plasmin activator, a neprilysin (NEP) activator, an IDE(insulin degrading enzyme) activator, a melatonin MT1 and/or MT2agonist, a TLX (tailless X receptor) ligand, a GluR1 ligand, a RAGE(receptor for advanced glycation end-products) antagonist, an EGFR(epidermal growth factor receptor) inhibitor, a FPRL-1 (formylpeptide-like receptor-1) ligand, a GABA antagonist or a MICAL (moleculeinteracting with casL) inhibitor such as an oxoreductase inhibitor, inassociation with a pharmaceutically acceptable carrier. In a furtherembodiment the present invention provides a combination comprising acompound of formula (I) or a pharmaceutically acceptable salt thereof,together with a further therapeutic agent as described herein above forsequential or simultaneous administration in separate or combinedpharmaceutical formulations.

In a further aspect, the invention provides a method of inhibitingproduction of amyloid-β protein and/or of treating or preventing aneurodegenerative disease, such as Alzheimer-type dementia (AD), Down'ssyndrome, cerebrovascular amyloid angiopathy (CAA), mild cognitiveimpairment (MCI), memory loss, presenile dementia, senile dementia,hereditary cerebral hemorrhage with amyloidosis, and other degenerativedementias such as dementias of mixed vascular and degenerative origin,dementia associated with supranuclear palsy, dementia associated withcortical basal degeneration, dementia associated with Parkinson'sDisease (PD), and dementia associated with diffuse Lewy Body type of AD,the method involving administering to a human subject suffering from thecondition a therapeutically or prophylactically effective amount of thepharmaceutical composition described above or of a compound of formula(I) as defined above, or a pharmaceutically acceptable salt thereof.“Effective amount” means an amount sufficient to cause a benefit to thesubject or at least to cause a change in the subject's condition.

Alzheimer's Disease (AD) is characterized pathologically by the presenceof neurofibrillary tangles (NFTs) and plaques, consisting of amyloid(Aβ) peptides of varying length, for example 42 amino acids (Aβ42) and40 amino acids (Aβ40). In addition to these pathological markers, brainatrophy is also evident. The build up of plaques is believed to be dueto the aggregation of Aβ peptides. Aβ peptides are formed in the brainby the sequential cleavage of amyloid precursor protein (APP) byβ-secretase (BACE-1) and γ-secretase. Therefore potential AD drugs aimedat inhibiting amyloid formation by inhibiting BACE-1 or γ-secretase,must be able to achieve adequate exposure in the brain, in order toexert an effect on AD.

Although BACE-1 represents an attractive target to halt or slow theproduction of amyloid peptides, various groups have found it challengingto identify BACE-1 inhibitors that can penetrate the central nervoussystem (CNS) and thus inhibit the enzyme at the site of action.

The brain is protected by several barriers including the blood brainbarrier (BBB) and transporters (Hitchcock and Pennington, J Med Chem2006, 29, 7559; Ueno, Curr. Med. Chem. 2007, 14, 1199; Gloor et al.,Brain Res. Rev. 2001, 36, 258). Several efflux transporters have beencharacterised which prevent compounds entering the brain. One of thebest characterised and most prominent in preventing the CNS penetrationof xenobiotics is P-glycoprotein (Pgp) (Kusuhara and Sugiyama, DrugDiscovery Today, 2001, 6, 150; Mahar Doan et al., J. Pharm. Expt. Ther.2002, 303, 1029; Lin, Drugs of Today 2004, 40, 5; Lin & Yamazaki, ClinPharmacokinet. 2003, 42, 59; Schinkel, Adv. Drug Deliv. Rev. 1999, 36,179). It has been shown that Pgp efflux is important for BACE-1inhibitors (Hussain et al., J. Neurochem. 2007, 100, 802). Thus,overcoming Pgp efflux is important.

Those skilled in the art will appreciate that there are several ways tomeasure or predict CNS penetration in vitro or in vivo. The potentialfor CNS penetration can be assessed in vitro by determining whether acompound can be subjected to Pgp efflux, i.e. by conducting an in vitroPgp assay. Those skilled in the art will appreciate that a number ofcell lines can be used and that these cell lines may or may not affectthe results of the assay. One such assay is described below (CyprotexUK).

The following MDR-1 MDCK assay was used to assess Pgp efflux. The assaywas conducted at Cyprotex Discovery Ltd. 15 Beech Lane, Macclesfield,Cheshire, UK, SK10 2DR

MDR1-MDCK Permeability (Bi-Directional; pH 7.4/pH 7.4)

Protocol Summary

MDCK cells are an epithelial cell line of canine kidney origin. Thesecells can be transfected to stably express active P-glycoprotein(MDR1-MDCK) and are ideal for studying drug efflux. Test compound wasadded to either the apical or basolateral side of a confluent monolayerof MDR1-MDCK cells and permeability was measured by monitoring theappearance of the test compound on the opposite side of the membraneusing LC-MS/MS. From this an apparent permeability (P_(app)) coefficientand efflux ratio was measured/calculated.

Objective

To measure the permeability of test compound in the apical tobasolateral (A-B) and basolateral to apical (B-A) direction acrossMDR1-MDCK cells. A ratio of B-A and A-B permeabilities was calculated(efflux ratio) to show whether the compound undergoes P-glycoproteinefflux.

Compounds were provided as a 200 μL solution of 10 mM test compound inDMSO.

Experimental Procedure

MDR1-MDCK cells obtained from the NIH (Rockville, Md., USA) were used.Following culture to confluency, the monolayers were prepared by rinsingboth basolateral and apical surfaces twice with pH 7.4 buffer at 37° C.Cells were then incubated with pH 7.4 buffer in both apical andbasolateral compartments for 40 min to stabilise physiologicalparameters.

Buffer at pH 7.4 was then removed from the apical compartment andreplaced with test compound dosing solutions. The solutions wereprepared by diluting 10 mM test compound in DMSO with buffer to give afinal test compound concentration of 10 μM (final DMSO concentrationadjusted to 1%). The fluorescent integrity marker Lucifer yellow wasalso included in the dosing solution. The apical compartment insertswere then placed into ‘companion’ plates containing fresh buffer at pH7.4. Analytical standards were made from dosing solutions.

For basolateral to apical (B-A) experiments the experiment was initiatedby replacing buffer in the inserts then placing them in companion platescontaining dosing solutions. Incubations were carried out in anatmosphere of 5% CO₂ with a relative humidity of 95% at 37° C. for 60minutes.

After the incubation period, the companion plate was removed and apicaland basolateral samples diluted for analysis by LC-MS/MS. Test compoundpermeability was assessed in duplicate. On each plate compounds of knownpermeability characteristics were run as controls.

Test and control compounds were quantified by LC-MS/MS cassette analysisusing a 5-point calibration with appropriate dilution of the samples.Cyprotex generic analytical conditions were used. The startingconcentration (C₀) was determined from the dosing solution and theexperimental recovery calculated from C₀ and both apical and basolateralcompartment concentrations.

The integrity of the monolayers throughout the experiment was checked bymonitoring Lucifer yellow permeation using fluorimetric analysis.Lucifer yellow permeation is low if monolayers have not been damaged. Ifa Lucifer yellow P_(app) value was above QC limits in one individualtest compound well, then an n=1 result was reported. If Lucifer yellowP_(app) values were above QC limits in both replicate wells for a testcompound, the compound was re-tested. If on repeat, high Lucifer yellowpermeation was observed in both wells then toxicity or inherentfluorescence of the test compound was assumed. No further experimentswere performed in this instance.

Data Analysis

The permeability coefficient for each compound (P_(app)) was calculatedfrom the following equation:P _(app)=(dQ÷dt)÷(C ₀ ×A)

Where dQ/dt is the rate of permeation of the drug across the cells, C₀is the donor compartment concentration at time zero and A is the area ofthe cell monolayer. C₀ was obtained from analysis of the dosing solutionat the start of the experiment.

In addition, an efflux ratio (ER) was calculated from mean A-B and B-Adata. This is derived from:ER=((P _(app)(B−A))÷((P _(app)(A−B))

Two control compounds were screened alongside the test compounds,propranolol (highly permeable) and prazosin (a substrate forP-glycoprotein).

Surprisingly, compounds from the present invention were found to displaya lower Pgp efflux ratio than compounds exemplified in WO2009/091016indicating that they have the potential to show higher CNS penetration.Data for selected examples are shown in Table 1 below.

TABLE 1 MDR-1 MDCK Pgp assay data Example Pgp ER Comparative Example 126.2 Comparative Example 2 16.6 Comparative Example 3 24.0 ComparativeExample 4 20.7 2 1.7 3 1.4 4 1.0 1 0.7 Comparative Example 5 4.7Comparative Example 6 4.5 10 0.6 5 1.5 6 1.0 7 0.8 8 1.7 9 1.2 11 1.0 120.8 13 1.7 15 1.6 16 1.1 17 1.4 18 1.1 Note: Example 14 is intentionallynot included.Comparative Examples 1 to 6 are covered by published Internationalpatent application WO2009/091016; Comparative Examples 1 to 4 arespecifically described in WO2009/091016 as Examples 32, 35, 54 and 73respectively.Comparative Examples 5 and 6 areN-(3-((4aS,5S,7aS)-2-amino-5-(fluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethoxypicolinamide,andN-(3-((4aS,5R,7aS)-2-amino-5-methyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethoxypicolinamiderespectively.

The data demonstrate that compounds of the present invention andspecific examples 1-13 and 15-18 have lower Pgp efflux and thereforepotentially higher CNS penetration than representative examples fromWO2009/091016 using the aforementioned recognized method of assessingCNS penetration. For example, Comparative Examples 1 to 6 have higherPgp efflux ratios than compounds of the present invention. Furthermore,Comparative Examples 5 and 6 have higher Pgp efflux ratios than a closeanalogue, Example 10 of the present invention, which clearlydemonstrates the beneficial effect the trifluoromethyl group on thetetrahydrofuran ring exerts on Pgp efflux, i.e. the trifluoromethylgroup reduces Pgp efflux.

Those skilled in the art will appreciate that the in vitro Pgp assaydescribed above is a predictive assay of in vivo CNS penetration. It isthus also highly desirable if the decreased Pgp-mediated effluxtranslates to the in vivo situation. Those skilled in the art willappreciate there are many ways to assess the CNS penetration ofcompounds in vivo. For example, one can quantify compound concentrationsin blood or plasma and brain and calculate a brain:blood (Br:Bl) orbrain:plasma (Br:Pl) ratio. This method has been used historically andhas been widely accepted as a method of determining CNS penetration(Summerfield et al., J Pharmacol. Expt. Ther. 2007, 322, 205). Thoseskilled in the art will appreciate that this type of assay could beconducted at steady state, a single time point, multiple time points orcould be done by quoting Area Under the Curve (AUC) ratios. All methodsare equally valid but each may have certain caveats that will beappreciated by those skilled in the art. Recent literature has beenpublished to suggest that it is important to consider the freeconcentrations in vivo and that when no efflux occurs from the brain thefree plasma concentration should be the same or equivalent to the freebrain concentration (Kalvass and Maurer, Biopharmaceutics & DrugDisposition 2002, 23, 327; Mauer et al, Drug Metab. Disposition 2005,33, 175; Trainor Expert Opin. Drug Discov. 2007, 2, 51). Thus, acompound that can freely penetrate the CNS and is not subjected toactive efflux, for example by Pgp or another transporter, shoulddemonstrate a free brain:free plasma (Br_(fr):Pl_(fr)) or an unboundbrain:unbound plasma (Br_(u):Pl_(u)) of approximately 1:1. Those skilledin the art will appreciate that the free or unbound concentrations canbe calculated by multiplying the total brain or total plasmaconcentration by the fraction unbound in brain tissue or plasma, whichcan be measured by the assay described below. Those skilled in the artwill appreciate that the fraction unbound may change with experimentalfactors, for example concentration, or temperature, etc. Those skilledin the art will be able to assess this and select the most appropriateset of conditions. Those skilled in the art will also appreciate that aslong as the conditions are the same for each compound screened then theassay will give consistent data for the range of compounds tested thusminimising any discrepancies. It has also been proposed that drugconcentrations in cerebrospinal fluid (CSF) are equivalent to free brainconcentrations for compounds which are not actively effluxed from thebrain (He et al., Xenobiotica 2009, 39, 687). Thus another method ofdetermining CNS penetration would be to assess the CSF:free plasma(CSF:Pl_(fr)) or CSF:unbound plasma (CSF:Pl_(u)). If the free drug inplasma is able to permeate into the CNS and is not actively influxed oreffluxed then the CSF:Pl_(fr) or CSF:Pl_(u) should be approximately 1:1.Those skilled in the art will appreciate the issues associated withdetermining CSF drug concentrations and extracting CSF, for example CSFcan be contaminated by blood depending on the method of withdrawal, alsothe CSF concentrations may be of lower accuracy, depending on the doseused.

Thus it has been shown that a BACE inhibitor from GlaxoSmithKline(GSK188909), BACE-1 IC₅₀ 5 nM, which has low CNS exposure wasineffective at lowering Aβ40 production in the brains of TASTPM mice(which overexpress both human APPswe^(K59N/M596I) and PS-1^(M146v)) uponacute administration (Hussain et al., J. Neurochem. 2007, 100, 802-809).Following an oral dose of 250 mg/kg the brain concentration of GSK188909in TASTPM mice was 0.62 uM. When a Pgp inhibitor (GF120918) was dosed 5hours before the oral administration of GSK188909, the brainconcentration of GSK188909 was found to be 5.43 uM following an oraldose of 250 mg/kg, i.e. the co-administration of a Pgp inhibitor causedan almost 9-fold increase in CNS penetration, showing Pgp efflux is animportant mechanism in preventing BACE inhibitors from penetrating theCNS. Furthermore, in the absence of a Pgp inhibitor, a 250 mg/kg oraldose of GSK188909 did not have any effect on brain Aβ40 levels in TASTPMmice, whereas when a Pgp inhibitor was co-administered (5 hours prior tothe administration of GSK188909) a 68% reduction in brain Aβ40 levelsrelative to vehicle treated mice was observed.

Another paper has reported a similar effect with three BACE-1 inhibitorsfrom Bristol-Myers Squibb (Meredith et al., J. Pharm. Expt. Ther. 2008,326, 502-513). The three reported compounds were found to be Pgpsubstrates in vitro. When dosed to mice, the three compounds showed lowCNS penetration and did not lower amyloid levels in the brain but wereable to lower plasma amyloid levels. When the same three compounds wereadministered to Pgp knockout (KO) mice, the level of CNS penetrationincreased and the compounds were able to lower amyloid levels in thebrain.

Researchers at Schering-Plough have also published papers (Iserloh etal., Bioorg. Med. Chem. Lett. 2008, 18, 418) to show that BACE-1inhibitors from their series (e.g. example 11 from the aforementionedreference), are subject to Pgp efflux, as a result of which the compoundwas found to display a low Br:Pl (<0.1) in the rat.

The literature cited above emphasises the difficulties in identifyingBACE-1 inhibitors which are not subjected to Pgp efflux. Such inhibitorswould be highly desirable and many research groups have attempted todiscover such compounds without success. Thus BACE-1 inhibitors whichare not Pgp substrates and can therefore readily penetrate the CNS andlower amyloid in the brain would be desirable.

More recently, researchers at Wyeth have reported extensive work toovercome Pgp efflux in a series of cyclic acylguanidine BACE-1inhibitors (Malamas et al, Bioorg. Med. Chem. Lett. 2010, 20, 6597).Compounds were discovered that were weak Pgp substrates and with Br:Plapproaching 1:1. However, two lead examples with reduced Pgp efflux (84and 89 from the aforementioned reference), did not lower Aβ40 in thebrain of Tg2576 mice 8 hours after a 30 mg/kg oral dose. The lack ofefficacy was attributed to the fact the compounds showed high braintissue binding. Thus, it is important to discover BACE-1 inhibitors thatare not Pgp substrates but still have a reasonable unbound fraction inbrain tissue and are able to lower amyloid in the brain.

It has also been shown that BACE inhibitors that are not Pgp substratesin vitro can penetrate the CNS (e.g. TC-1 from Merck), and can lowerAβ40 levels in the brain of APP-YAC mice and monkeys (Sankaranarayananet al., J. Pharmacol. Expt. Ther. 2009, 328, 131-140). Thus, in vitroPgp assays showed TC-1 not to be a Pgp substrate and when TC-1 was dosedto APP-YAC mice (100 mg/kg i.p.) it was able to modestly penetrate theCNS as shown by the brain concentrations and the brain:plasma ratio andthis ability resulted in moderate lowering of brain amyloid.

Plasma conc. Brain conc. Reduction in brain Time (μM) (μM) Br:Pl Aβ40(%) 2 h 25 1.6 0.06 26 4 h 13 1.8 0.14 29 Brain and plasma concentrationof TC-1 following 100 mg/kg i.p. dose and corresponding effects on brainAb40 levels, in APP-YAC mice.

In separate experiments it was shown that TC-1 could penetrate the CSFof monkeys when co-administered with a CYP3A4 inhibitor (ritonavir). Inthese experiments the average plasma concentration of TC-1 was found tobe 2.7 uM, whilst the CSF concentration was found to be 0.025 uM.However, as TC-1 is ˜99% bound to plasma proteins the free plasmaconcentration was calculated to be ˜0.027 nM. It was found that CSF Aβ40levels showed a 42% decrease relative to a vehicle treated controlgroup. Thus, a BACE inhibitor that can freely penetrate the CNS would beexpected to be able to lower amyloid levels in the CNS. It would bebeneficial not to have to be co-dosed with a CYP3A4 inhibitor.

The compounds of the present invention have been shown to lower Aβproduction in cellular assays which correlates with their ability tolower Aβ production in animals. Thus, the compounds of the presentinvention will have utility in lowering Aβ production in humans and thuswill be useful in the treatment of neurodegenerative diseases such asAlzheimer's disease.

Rat In Vivo CNS Penetration

Male Sprague Dawley rats were acquired from Charles River UK Ltd.(Margate, UK) and housed according to UK Home Office guidelines. Drugswere made up to the appropriate concentrations in 0.5% methyl cellulose.Animals were dosed orally (2 mL/kg) by gavage at the doses outlined inTables 2 to 4 below.

At the time points post-dosing, specified in Tables 2 to 4 below theanimals were administered an i.p. injection of sodium pentobarbitone(approximately 330 mg/kg for terminal anaesthesia).

Using a guillotine, the animals were decapitated and trunk bloodcollected into 15 ml Falcon tubes containing 100 IU heparin. Blood wasvortexed followed by centrifugation at 6000 rpm, 4° C. for 5 minutes.Plasma was collected for DMPK and ELISA assays and stored at −80° C.until use. Brains were dissected out and divided along the midline,weighed and stored at −80° C. until further use.

Method for Analysis of Plasma, Brain and CSF Samples

Preparation of Acetonitrile Working Solutions

Test compound was prepared as a 1 mg free base/mL solution in DMSO,vortexed and sonicated for 5 min. The 1 mg/mL DMSO solution was dilutedto 10 and 30 μg/mL acetonitrile stocks, by adding 10 μL to 990 μLacetonitrile and 30 μL to 970 μL acetonitrile, respectively. The 10 and30 μg/mL acetonitrile stocks were then serially diluted 1:9 (v/v) (100μL stock into 900 μL acetonitrile) to give the following solutions:0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg/mL acetonitrile.

Preparation of Plasma Standards, Blanks and Samples

Control male Sprague Dawley rat plasma and the study plasma samples werestored at −80° C. until the day of analysis when they were thawed atroom temperature. Control plasma was centrifuged (2,000 g for 10 min)and aliquoted (90 μL) into eppendorf tubes for preparation of standardsand blank samples. Study samples were previously aliquoted (100 μL) intoeppendorf tubes immediately following collection of the plasma.

An aliquot (10 μL) of the appropriate acetonitrile stock was added tothe control plasma (to give a final volume of 100 μL) to give therequired calibration standards covering the range 1-3000 ng/mL. Doubleblank and blank samples were prepared by adding 10 μL of acetonitrile to90 μL of blank plasma.

Preparation of Brain Standards, Blanks and Samples

Control male Sprague Dawley rat brain and the study brain samples wereweighed after collection and stored at −80° C. until the day of analysiswhen they were thawed at room temperature. Once thawed brains werediluted with water (4 mL per gram of tissue) and homogenised using amechanical homogeniser. An aliquot (100 μL) of each study sample wastaken into Micronics tubes ready for analysis and sufficient aliquots(90 μL) of control brain homogenate prepared for preparation ofstandards and blanks.

An aliquot (10 μL) of the appropriate acetonitrile stocks was added tothe control brain homogenate (to give a final volume of 100 μL) to givethe required calibration standards covering the range 1.5-5000 ng/g.Double blank and blank samples were prepared by adding 10 μL ofacetonitrile to 90 μL of blank brain homogenate.

Extraction of Plasma and Brain Samples, Standards and Blanks

Each plasma and brain homogenate sample, standard and blank (100 μL) wasextracted with an aliquot (300 μL) of acetonitrile (containing 0.1%formic acid and 100 ng/mL of an appropriate internal standard). Doubleblanks were extracted with an aliquot (300 μL) of acetonitrilecontaining 0.1% formic acid). All samples, standards and blanks werethen vortex mixed and centrifuged (2000 g for 15 min). An aliquot (50μL) of the resulting supernatant was then taken into a 2 mL 96-deep wellplate and diluted with acetonitrile:water (50:50 v/v) (150 μL) ready foranalysis by a specific LC-MS/MS method.

Preparation of CSF Samples, Standards and Blanks

Control male Sprague Dawley rat CSF and the study CSF samples werestored at −80° C. until the day of analysis when they were thawed atroom temperature. An aliquot (50 μL) of each study sample was taken intoMicronics tubes ready for analysis and sufficient aliquots (45 μL) ofcontrol CSF prepared for preparation of standards and blanks.

An aliquot (5 μL) of the appropriate acetonitrile stocks was added tothe control CSF (to give a final volume of 50 μL) to give the requiredcalibration standards covering the range 1-1000 ng/mL. Double blank andblank samples were prepared by adding 5 μL of acetonitrile to 45 μL ofblank CSF.

Extraction of CSF Samples, Standards and Blanks

Each CSF sample, standard and blank (50 μL) was extracted with analiquot (150 μL) of acetonitrile (containing 0.1% formic acid and 100ng/mL of an appropriate internal standard). Double blanks were extractedwith an aliquot (150 μL) of acetonitrile containing 0.1% formic acid.All samples were then vortex mixed and an aliquot (50 μL) of each wasthen further diluted in 150 μL of acetonitrile:water (50/50 v/v) in a 2mL 96-deep well block ready for LC-MS/MS analysis. All samples were thenanalysed using a Waters Acquity UPLC coupled to a Waters Xevo TQ massspectrometer.

LC Conditions:

Column. Acquity UPLC BEH C18, 1.7 um, 2.1×50 mm, maintained at 40° C.

Mobile Phase: A=95% Water:5% MeOH containing 0.01M Ammonium acetate

-   -   B=5% Water:95% MeOH containing 0.01M Ammonium acetate        Gradient:

Time (min) B (%) 0 5 1.2 95 1.5 95 1.7 5 2.0 5Flow rate^(.) 0.6 mL/min; injection volume 5 μL; autosampler temperature6° C.

LC flow was diverted to waste for the first 0.3 min of each injection

MS/MS transitions were optimised automatically by Waters QuanOptimisesoftware.

Amyloid Detection

DEA/NaCl Extraction of Aβ Peptides from Rat Brain:

100 ml of chilled 0.2% diethyl amine (DEA) in 50 mM NaCl (pH 10) wasfreshly prepared and 1 ml/25 mg brain tissue was added to eachhemisphere (i.e. 40× brain volume). The brains were immediatelyhomogenized using a Polytron PT 1200 for 1.5 minutes and samples left toincubate on ice for one hour after homogenisation. 3 ml of thehomogenate was transferred to a polyallomer tube (Beckman #362333) andspun at 133000×g (55,000 rpm) for 45 min at 4° C. The supernatant wasthen neutralised to pH 8-8.3 by adding 1/10 volume 0.5M Tris/HCl, pH6.8. The samples can be used fresh or snap frozen on dry-ice and storedat −80° C. until required for analysis

Human/Rat βAmyloid (40) ELISA (Wako Kit)

The Wako Aβ40 ELISA kit (Code No. 294-62501) uses the monoclonalantibody BNT77, raised against epitope Aβ(11-28) and the monoclonalantibody BA27, which specifically detects the C-terminal portion ofAβ40. This kit is used for the quantitative determination of human orrat Aβ(1-40) and also N-terminally truncated Aβ40 species (Aβ(x-40)) inbiological matrices such as tissue culture medium, tissue homogenate,CSF and plasma.

For analysis, plasma and brain samples are diluted 1:1 with the standarddiluent contained in the kit and CSF samples are diluted 1:8 with thestandard diluent contained in the kit. The assay is carried outaccording to manufacturers instructions and samples are analysed induplicate. Data is analysed using Microsoft Excel 2003 and statisticalanalysis is carried out using Genstat 9^(th) Edition.

Thus, when Comparative Example 4 was administered at a dose of 10 mg/kgp.o. and plasma, brain and CSF samples were collected 2, 4, 6 and 8hours post-dose the following concentrations were measured (Table 2):

TABLE 2 Data for Comparative Example 4 Time [Pl] ¹[Pl_(u)] [Br]²[Br_(u)] [CSF] (h) (nM) (nM) (nM) (nM) (nM) Br_(tot):Pl_(tot)Br_(u):Pl_(u) CSF:Pl_(u) 2 1257 440 971 65 104 0.8 0.15 0.24 4 1162 407874 59 88 0.7 0.14 0.22 6 834 292 570 38 63 0.7 0.13 0.22 8 484 169 36825 26 0.8 0.15 0.15 ¹Calculated by multiplying the [Pl] by Pl Fu.²Calculated by multiplying the [Br] by Br Fu.

From the above study, Comparative Example 4 showed a 59% and 64%reduction of Aβ40 in the brain at 4 and 6 hours respectively; and a 76%and 70% reduction of Aβ40 in the CSF at 4 and 6 hours respectively.

Certain compounds of the present invention have been assessed in vivo inthe rat to corroborate the levels of CNS penetration; these data arepresented in the tables below.

Surprisingly, it has been found that compounds of the present inventionshow increased CNS penetration in the rat relative to compounds fromWO2009/091016 by any of the aforementioned recognized methods ofdetermining CNS penetration. Thus, the compounds of the presentinvention may show improved profiles in that they more readily targetthe site of action, the brain, and therefore may show improved efficacyor efficacy at lower concentrations or doses or decreased peripherallymediated side effects, by way of preferential CNS partitioning, or acombination of any or all of these aspects.

Thus, when Example 8 of the present invention was administered at a doseof 10 mg/kg p.o. and plasma, brain and CSF samples were collected 2, 4,6 and 8 hours post-dose the following concentrations were measured(Table 3):

TABLE 3 Data for Example 8 Time [Pl] ¹[Pl_(u)] [Br] ²[Br_(u)] [CSF] (h)(nM) (nM) (nM) (nM) (nM) Br_(tot):Pl_(tot) Br_(u):Pl_(u) CSF:Pl_(u) 21189 124 3961 87 65 3.3 0.7 0.5 4 657 68 2582 57 39 3.9 0.8 0.6 6 229 24845 19 12 3.7 0.8 0.5 8 186 19 709 16 ³n.q. 3.8 0.8 ⁴n.d. ¹Calculated bymultiplying the [Pl] by Pl Fu. ²Calculated by multiplying the [Br] by BrFu. ³Not quantifiable. Concentrations close to or below the lower limitof quantification and could not be accurately quantified. ⁴Notdetermined.

From the above study, Example 8 showed a 68% and 72% reduction of Aβ40in the brain at 4 and 6 hours respectively; and an 82% and 74% reductionof Aβ40 in the CSF at 4 and 6 hours respectively. Thus compounds of thepresent invention show decreased Pgp efflux relative to previousdisclosures whilst demonstrating efficacy in the CNS. The efficacy isthus achieved with lower circulating plasma concentrations.

When Example 1 of the present invention was administered at a dose of 10mg/kg p.o. and plasma, brain and CSF samples were collected 2, 4, 6 and8 hours post-dose, the following concentrations were measured (Table 4):

TABLE 4 Data for Example 1 Time [Pl] ¹[Pl_(u)] [Br] ²[Br_(u)] [CSF] (h)(nM) (nM) (nM) (nM) (nM) Br_(tot):Pl_(tot) Br_(u):Pl_(u) CSF:Pl_(u) 2462 23 2338 30 23 5.1 1.3 1.0 4 298 15 1550 20 18 5.2 1.3 1.2 6 401 201492 19 10 3.7 1.0 0.5 8 228 11 1194 16 18 5.2 1.5 1.6 ¹Calculated bymultiplying the [Pl] by Pl Fu. ²Calculated by multiplying the [Br] by BrFu.

From the above study, Example 1 showed a 64% and 70% reduction of Aβ40in the brain at 4 and 6 hours respectively; and a 80% and 85% reductionof Aβ40 in the CSF at 4 and 6 hours respectively. Thus compounds of thepresent invention show decreased Pgp efflux relative to previousinventions whilst demonstrating efficacy in the CNS. The efficacy isthus achieved with lower circulating plasma concentrations.

Method For Determination of Plasma Protein Binding (PPB) and BrainTissue Binding (BTB)

Compound Preparation

Compounds were dissolved in DMSO to give a 1 mg free base/mL solution,before further dilution to 100 μg/mL in acetonitrile (100 μL of 1 mg/mLinto 900 μL acetonitrile).

Matrix Preparation

On the morning of dialysis, control male Sprague Dawley rat plasma andbrain, previously stored at −80° C. were thawed at room temperature.Plasma was checked for pH and if necessary adjusted to 7.4 with 1M HCl.Plasma was then centrifuged (2000 g for 10 min) and the brains dilutedwith 2 mL of Phosphate Buffered Saline (pH 7.4) per gram of tissue andhomogenised using a mechanical homogeniser. An aliquot (10 μL) of the100 μg/mL acetonitrile compound solution was then added to 1 mL ofplasma and brain homogenate and vortex mixed to give a final compoundconcentration of 1 μg/mL in matrix.

RED Plate Preparation

The Rapid Equilibrium Dialysis (RED) plate (Thermo Scientific) wasprepared in accordance with the manufacturers guidelines i.e. the baseplate was soaked in 20% (v/v) ethanol for 10 min and then rinsed twicewith deionised water before being allowed to dry. The base plate wasthen filled with the appropriate number of disposable inserts (n=3 percompound) (Thermo Scientific) and matrix containing 1 μg/mL compoundadded into the matrix chamber of the inserts (200 μL) and an aliquot(350 μL) of PBS added to the buffer chamber. The plate was then coveredwith an adhesive and incubated in air at 37° C. for 6 h with 130 rpmagitation.

Sampling

Following the 6 h incubation, the seal was removed and an aliquot (50μL) taken from the PBS chambers and dispensed into Micronics tubes.Also, an aliquot (50 μL) was removed from the matrix chambers and placedinto separate Micronics tubes. Plasma and brain was then matrix matchedwith 50 μL of drug-free PBS and the PBS samples with 50 μL of thecorresponding drug-free matrix, to give equal final compositions andvolumes (100 μL).

Sample Analysis

Samples were vortex mixed and an aliquot (300 μL) of acetonitrilecontaining 0.1% formic acid and 100 ng/mL of an appropriate internalstandard added. Samples were then mixed and centrifuged (2000 g for 15min) and an aliquot of the supernatant (100 μL) removed into a 96-deepwell plate and diluted with an equal volume of water ready for analysisby LC-MS/MS. The following data was obtained for the following compoundsin the above assay (Table 5).

TABLE 5 Compound Rat PPB (%) Rat Plasma fu Rat BTB (%) Rat Brain fuComparative 65.0 0.350 93.3 0.067 Example 4 Example 1 95.1 0.049 98.70.013 Example 8 89.6 0.104 97.8 0.022 Data represents the Mean of n = 3replicates fu = fraction unbound

From the data presented herein above it will be apparent to thoseskilled in the art that the compounds of Examples 1 and 8 achieve asimilar reduction of brain Aβ40 to that of Comparative Example 4, butwith a lower plasma concentration and free plasma concentration. This isadvantageous and indicates that the compounds of the invention will havesimilar or better efficacy at lower concentrations than the compounds ofWO2009/091016, and consequently will be less likely to cause unwantedperipherally mediated side effects, such as cardiovascular effects,phospholipidosis, liver toxicity, renal toxicity and gastrointestinaltoxicity.

Assessment of Effects on QTc Interval in Guinea Pigs

Male Dunkin-Hartley guinea pigs were weighed and anaesthetised using 4%isoflurane in carbogen. Anaesthesia was maintained at 1.5% isofluraneand the animals were kept under anaesthesia for the duration of thestudy. Xylazine at 2 mg/kg i.m. was administered into the hind limb as abradycardic agent to enable detection of QTc prolongation by thesoftware.

The carotid artery and jugular vein were cannulated with linescontaining heparinised saline and a 3-lead ECG connected and monitoredusing LabChart Pro software. Animals were allowed to stabilise for 30minutes after the completion of the surgical procedure, before theinitiation of an i.v. vehicle (5% DMSO/90% MilliQ/5% 0.1N HCl) infusionfrom time zero (infusion rate=0.2 ml/kg/min). At 10 mins, an arterialblood sample was collected for PK analysis (150u1; all collectionsyringes were heparinised). At 12 mins, drug infusion was started @ 2.0mg/kg/10 min i.v. The dose was increased to 6.0 mg/kg/10 min, then 20mg/kg/10 min i.v., with a 10 minutes infusion period and two minutesblood sampling at each dose. After the final dose, a blood sample wastaken and a second vehicle infusion initiated. Eight minutes later aterminal blood sample was collected for plasma PK analysis and theanimal killed by a Schedule 1 method.

QTc (Bazett's) changes were analysed using LabChart Pro software. QTcwas unchanged up to the highest dose/concentration tested, whichcorresponded to an unbound plasma concentration of 9503 nM for Example 8and 296 nM for Example 1.

Assessment of Effects on QTc Interval in Beagle Dogs

Male beagle dogs were weighed and injected with sodium thiopental forinduction of anesthesia. Anaesthesia was maintained with mixture of1-1.5% isoflurane and oxygen and the animals were kept under artificialrespiration and anesthesia using isoflurane for the duration of thestudy.

The carotid artery and saphenous vein were cannulated with linescontaining heparinised saline and a LII ECG connected and monitoredusing polygraph system. Animals were allowed to stabilise for 30 minutesbefore the infusion of compound solution and the infusion via cannulawas started from time zero with 1 mg/kg/10 min. The dose was increasedto 3 mg/kg/10 min, then 10 mg/kg/10 min. An arterial blood sample wascollected after every dosing for PK analysis.

QTc was unchanged up to the highest dose/concentration tested, whichcorresponded to an unbound plasma concentration of 4128 nM for Example 8and 1329 nM for Example 1.

Next, methods for preparing the compound of the formula (I) or apharmaceutically acceptable salt thereof according to the presentinvention will be described.

A. General Preparation Method A:

In the formula, X, Y and A are as defined above.

General Preparation Method A is a method for preparing a compound A-(15)which corresponds to compound (I) according to the present inventionfrom a compound A-(1) as a raw material through multiple steps of StepA-(i) to Step A-(xiv).

The compound A-(1) is commercially available. Step A-(i):

This step is a step of obtaining a compound A-(2) by opening the epoxideA-(1) with a sulfonium ylide to generate an intermediate allylicalkoxide which is then alkylated to give the compound A-(2). Thoseskilled in the art will appreciate that this transformation can beconducted in one pot or as two individual reactions. Those skilled inthe art will appreciate the benefits and drawbacks of a one pot reactioncompared to conducting two separate reactions and choose the best methodfor their requirements accordingly.

Specifically, the epoxide A-(1) can be opened by the anion oftrimethylsulfonium iodide and resultant loss of dimethylsulfide to givethe corresponding allylic alkoxide. Trimethylsulfonium iodide can bedeprotonated with a suitable base, for example butyl lithium. Thesolvent used in the reaction is not particularly limited insofar as itdoes not interfere with the reaction. Examples of suitable solventsinclude THF. Those skilled in the art will appreciate that the wordsolvent in this instance is used to denote the liquid in which thereaction is effected and that the reagents may not be dissolved.Preferably the reaction should be conducted below room temperature,preferably −30-20° C. Upon addition the reaction may be warmed to roomtemperature to facilitate reaction. The reaction time is notparticularly limited and is usually 5 minutes to 24 hours, preferably1-6 hours.

Those skilled in the art will appreciate that the alkoxide generatedfrom this reaction can be reacted with an alkylating agent directly,such as tert-butyl bromoacetate, and that this reaction may proceed withor without additional solvents. If additional solvents are required tofacilitate reaction, then solvents such as DMF or NMP are suitable. Thereaction temperature is not particularly limited. Suitable reactiontemperatures include room temperature to 80° C., preferably roomtemperature. The reaction time is not particularly limited and isusually 5 minutes to 1 week, preferably 1-48 hours.

Those skilled in the art will appreciate that the intermediate alkoxidecould be quenched, isolated and purified then subjected to independentalkylation conditions. This reaction can be performed under the sameconditions as those usually used in O-alkylation reaction of an alcoholcompound (such as the conditions described in Tetrahedron Lett. 46(2005) 45, 7751-7755). In this reaction, the compound A-(2) can beobtained by adding a base such as sodium hydride to a solution of theintermediate alcohol in THF to prepare an alkoxide, and then reactingthe alkoxide with the tert-butyl bromoacetate, for example. The solventused in the reaction is not particularly limited insofar as it does notinhibit the reaction and allows the starting material to be dissolvedtherein to a certain extent. Examples of the solvent include solventssuch as THF, DMF and dimethyl sulfoxide. The reaction can be performedby causing 1 to 3 equivalents of an appropriate base to act in thepresence of such a solvent. Examples of the base used include sodiumhydride, potassium hydride and t-butoxypotassium. The reaction time isnot particularly limited and is usually 0.5 to 72 hours, and preferably0.5 to 12 hours. The reaction temperature is usually −20° C. to 50° C.

A more preferable result such as an improved yield may be achieved byadding a salt such as tetrabutylammonium iodide in this reaction.

Step A-(ii):

This step is a two step sequential reaction to obtain compound A-(3)from compound A-(2) by deprotecting the ester group then forming aWeinreb amide.

Specifically, the tert-butyl ester of compound A-(2) can be deprotectedunder the same conditions as those generally used in deprotection of atert-butyl ester compound (such as the conditions described in adocument such as T. W. Greene and P. G. M. Wuts, “Protective Groups inOrganic Chemistry, Third Edition”, John Wiley & Sons (1999), p.404-408). In this reaction, the compound A-(2) can be reacted with anappropriate acid in a suitable solvent, such as formic acid, as solventand acid, for example. The solvent used in the reaction is notparticularly limited insofar as it does not inhibit the reaction andallows the starting material to be dissolved therein to a certainextent. The reaction time is not particularly limited and is usually 0.5to 72 hours, and preferably 0.5 to 24 hours. The reaction temperature isusually ice-cold temperature to 60° C.

The intermediate acid can then be transformed to the Weinreb amide(Tetrahedron Lett. 1981, 22, 3815) by reaction ofN,O-dimethylhydroxylamine hydrochloride under standard amide formationconditions, ie by condensing the intermediate acid withN,O-dimethylhydroxylamine hydrochloride using a condensing agent.Alternatively, this step is a step of obtaining a compound A-(3) bycondensing the intermediate acid with N,O-dimethylhydroxylaminehydrochloride by acylation reaction.

The condensation reaction of the intermediate acid withN,O-dimethylhydroxylamine hydrochloride using a condensing agent can beperformed under the same conditions as those usually used and describedin the following documents. Examples of the known method include thosein Rosowsky et al.; J. Med. Chem., 34 (1), 227-234 (1991), Brzostwska etal.; Heterocycles, 32 (10), 1968-1972 (1991), and Romero et al.; J. Med.Chem., 37 (7), 998-1014 (1994).

The N,O-dimethylhydroxylamine hydrochloride may be a free form or asalt.

The solvent in this reaction is not particularly limited insofar as itdoes not inhibit the reaction. Examples of the solvent includetetrahydrofuran, 1,4-dioxane, ethyl acetate, methyl acetate,dichloromethane, chloroform, N,N-dimethylformamide, toluene,acetonitrile and xylene. Examples of the condensing agent include CDI(N,N′-carbonyldiimidazole), Bop(1H-1,2,3-benzotriazol-1-yloxy(tri(dimethylamino))phosphoniumhexafluorophosphate), WSC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride), DCC (N,N-dicyclohexylcarbodiimide), diethylphosphorylcyanide, PyBOP (benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate) and EDC.HCl(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). Suitableconditions include an agent to activate the acid, such as N,N′-carbonyldiimidazole. One equivalent to a large excess ofN,O-dimethylhydroxylamine hydrochloride is used with respect to theintermediate acid. One equivalent to a large excess of an organic basesuch as triethylamine may be added where necessary.

The reaction time is not particularly limited and is usually 0.5 to 72hours, and preferably 0.5 to 24 hours. The reaction temperature variesaccording to the raw material used, the solvent and the like and is notparticularly limited. Ice-cold temperature to solvent reflux temperatureis acceptable, ice cold to room temperature is preferable.

Step A-(iii):

This step is a step of obtaining a compound A-(4) by reaction of anorganometallic (aryllithium reagent or a Grignard reagent) reagent withcompound A-(3) as described in Tetrahedron Lett. 1981, 22, 3815.

The reaction in this step can be performed under the same conditions asthose described in Tetrahedron Lett. 1981, 22, 3815, for example.

The aryllithium reagent (including heterocyclic) or the Grignard reagent(including heterocyclic) can be prepared by a method known to a personskilled in the art. Specifically, the corresponding phenyl lithiumreagent or phenyl magnesium (Grignard) reagent can be prepared byhalogen-metal exchange between an aryl halide compound and acommercially available organometallic reagent such as an alkyllithiumreagent such as n-, sec- or tert-butyllithium or a Grignard reagent suchas isopropylmagnesium bromide, or metallic magnesium, for example.

The solvent used in this step varies according to the starting materialand the reagent used, and is not particularly limited insofar as it doesnot inhibit the reaction, allows the starting material to be dissolvedtherein to a certain extent, and is always inert during the reaction.Preferable examples of the solvent include organic solvents such asdiethyl ether, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane,benzene and toluene, and mixed solvents thereof. The reaction time isnot particularly limited and is usually 0.1 to 48 hours, and preferably0.1 to 12 hours. The reaction temperature varies according to thestarting material, the reagent used and the like, and is preferablymaintained to be low, for example, at −78-−60° C.

Step A-(iv):

This step is a step of obtaining a compound A-(5) by oximation of thecompound A-(4).

The reaction in this step can be performed under the same conditions asthose usually used in oximation reaction of a carbonyl compound such asthe conditions described in Org. Lett. 9 (2007) 5, 753-756, Tetrahedron:Asymmetry 5 (1994) 6, 1018-1028 and Tetrahedron 54 (1998) 22, 5868-5882.

Specifically, the compound A-(5) can be obtained by reacting thecompound A-(4) with hydroxylamine or a hydroxylamine salt (such ashydroxylamine hydrochloride or hydroxylamine sulfate) in the presence ofa base or in the absence of a base, for example.

The solvent used in this reaction is not particularly limited insofar asit does not inhibit the reaction. Preferable examples of the solventinclude organic solvents such as ethanol, methanol, tetrahydrofuran,1,4-dioxane, 1,2-dimethoxyethane and dichloromethane, and mixtures ofthese solvents and water. Examples of the base used include sodiumacetate, pyridine, sodium hydroxide, cesium hydroxide, barium hydroxideand 2,6-lutidine. The reaction time is not particularly limited and isusually 5 minutes to 24 hours, and preferably 5 minutes to 12 hours. Thereaction temperature is usually −20° C. to solvent reflux temperature,and more preferably 0° C. to solvent reflux temperature.

Step A-(v):

This step is a step of obtaining a compound A-(6) by a thermalintramolecular cycloaddition of the alkenyl oxime A-(5).

The reaction is conducted in the presence of an additive, for examplehydroquinone.

The solvent used in this reaction is not particularly limited insofar asit does not inhibit the reaction. Suitable reaction solvents includehigh boiling solvents such as xylenes. The reaction temperature is notparticularly limited and is usually 80-200° C. or solvent refluxtemperature. The reaction time is not particularly limited and isusually 0.5 to 48 hours, and preferably 0.5 to 24 hours.

Step A-(vi):

This step is a step of obtaining a compound A-(7) by subjecting thecompound A-(6) to reductive cleavage reaction of the N—O bond.

The reductive cleavage reaction of the N—O bond can be performed underthe conditions using zinc-acetic acid, a metal catalyst such ashydrogen-platinum oxide, or lithium aluminum hydride, for example.

The reaction using zinc such as zinc-acetic acid can be performed underthe same conditions as those described in J. Org. Chem. 2003, 68,1207-1215 and Org. Lett. 7 (2005) 25, 5741-5742, for example. Examplesof the acid used include acetic acid, formic acid and hydrochloric acid.The solvent used in the reaction is not particularly limited insofar asit does not inhibit the reaction and allows the starting material to bedissolved therein to a certain extent. Examples of the solvent includemethanol, ethanol, 1,4-dioxane, THF and water. The above acid may alsobe used as a solvent. The reaction temperature is usually −20° C. tosolvent reflux temperature, and preferably ice-cold temperature tosolvent reflux temperature. The reaction time is not particularlylimited and is usually 5 minutes to 48 hours, and preferably 5 minutesto 24 hours.

The reaction using a metal catalyst such as hydrogen-platinum oxide canbe performed under the same conditions as those described inTetrahedron: Asymmetry 5 (1994) 6, 1018-1028 and Tetrahedron, Vol. 53,No. 16, pp 5752-5746, 1997, for example. The compound A-(7) can beobtained by hydrogenating the compound A-(6) using platinum oxide as acatalyst in a solvent such as methanol, for example.

The reaction using lithium aluminum hydride can be performed under thesame conditions as those described in Bull. Chem. Soc. Jpn., 66,2730-2737 (1993), for example. The compound A-(7) can be obtained byreducing the compound A-(6) using lithium aluminum hydride in a solventsuch as ether, for example.

Step A-(vii):

This step is a step of obtaining a compound A-(8) from the compoundA-(7). The thiourea derivative A-(8) can be obtained from the compoundA-(7) by a method known to a person skilled in the art.

The compound A-(8) can be obtained in this step by reacting the compoundA-(7) with benzoyl isothiocyanate in a solvent such as dichloromethaneor toluene. This reaction can be performed under the same conditions asthose described in J. Med. Chem. 1990, 33, 2393-2407, for example. Thesolvent used in the reaction is not particularly limited insofar as itdoes not inhibit the reaction and allows the starting material to bedissolved therein to a certain extent. Examples of the solvent includedichloromethane, chloroform, toluene, 1,4-dioxane and THF. The reactiontemperature is usually −20° C. to solvent reflux temperature, andpreferably ice-cold temperature to solvent reflux temperature. Thereaction time is not particularly limited and is usually 5 minutes to 48hours, and preferably 5 minutes to 24 hours.

Step A-(viii):

This step is a method of obtaining a compound A-(9) by cyclizing thecompound A-(8).

In this reaction, the compound A-(8) can be cyclized under variousconditions to obtain the compound A-(9) by activating the alcohol ofcompound A-(8).

For example, the compound A-(9) can be obtained in this reaction byheating the compound A-(8) in a solvent such as methanol in the presenceof an acid such as concentrated hydrochloric acid, for example. Thesolvent used in the reaction is not particularly limited insofar as itdoes not inhibit the reaction and allows the starting material to bedissolved therein to a certain extent. Examples of the solvent includesolvents such as methanol, ethanol, 1-propanol and water, mixed solventsthereof, and acids used as a solvent. The reaction can be performed byusing one equivalent to a large excess of an appropriate acid to act inthe presence or absence of such a solvent. Examples of the acid usedinclude concentrated hydrochloric acid, hydrobromic acid, sulfuric acid,trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonicacid and mixtures thereof. The reaction time is not particularly limitedand is usually 0.5 to 72 hours, and preferably 0.5 to 24 hours. Thereaction temperature is usually ice-cold temperature to solvent refluxtemperature.

Alternatively, the compound A-(9) can be obtained by reacting thecompound A-(8) with trifluoromethanesulfonic anhydride in a solvent suchas dichloromethane in the presence of a base such as pyridine. Thesolvent used in the reaction is not particularly limited insofar as itdoes not inhibit the reaction and allows the starting material to bedissolved therein to a certain extent. Those skilled in the art willappreciate that a solvent is not always required and the reaction mayalso be conducted in the absence of a solvent, for example when the baseis pyridine. Examples of the solvent include solvents such asdichloromethane, chloroform, 1,2-dichloroethane, THF,1,2-dimethoxyethane and toluene, and mixed solvents thereof. Thereaction can be performed using 1 to 20 equivalents of an appropriatebase in such a solvent. Examples of the base used include pyridine,2,6-lutidine, sodium carbonate, potassium carbonate and mixturesthereof. The reaction time is not particularly limited and is usually0.5 to 24 hours, and preferably 0.5 to 12 hours. The reactiontemperature is usually −78° C. to room temperature.

Step A-(ix):

This step is a method of obtaining the compound A-(10) by deprotectingthe protecting group of the compound A-(9). The compound A-(10) can beobtained under deprotection conditions known to a person skilled in theart.

When the protecting group is a benzoyl group, the compound A-(10) can beobtained in this reaction by heating the compound A-(9) in a solventsuch as methanol in the presence of a base such as DBU, for example.This reaction can be performed under the same conditions as thosedescribed in Synth. Commun. 32 (2), 265-272 (2002), for example. Thesolvent used in the reaction is not particularly limited insofar as itdoes not inhibit the reaction and allows the starting material to bedissolved therein to a certain extent. Examples of the solvent includesolvents such as methanol, ethanol and 1-propanol. The reaction can beperformed using 1 to 20 equivalents of an appropriate base in such asolvent. Examples of the base used include DBU. The reaction time is notparticularly limited and is usually 0.5 to 24 hours, and preferably 0.5to 12 hours. The reaction temperature is usually room temperature tosolvent reflux temperature.

Alternatively, compound A-(10) can be obtained in this reaction byheating compound A-(9) with an inorganic base such as potassiumcarbonate, for example. The solvent used in the reaction is notparticularly limited insofar as it does not inhibit the reaction andallows the starting material to be dissolved therein to a certainextent. Examples of the solvent include solvents such as methanol,ethanol and 1-propanol. The reaction can be performed using 1 to 20equivalents of an appropriate base in such a solvent, and preferably aslight excess is used. Examples of the base used include potassiumcarbonate. The reaction time is not particularly limited and is usually0.5 to 24 hours, and preferably 0.5 to 12 hours. The reactiontemperature is usually room temperature to solvent reflux temperature,and preferably 50-100° C. Those skilled in the art will appreciate thatthe selected solvent will limit the reaction temperature by its refluxtemperature. Examples of suitable solvents include refluxing methanol.

Step A-(x):

This step is a step of obtaining the compound A-(11) by nitrationreaction of the compound A-(10). In this nitration reaction, thecompound A-(11) can be obtained from the compound A-(10) by a methodknown to a person skilled in the art. Examples of the nitrating agentused in the reaction include potassium nitrate/concentrated sulfuricacid, fuming nitric acid/concentrated sulfuric acid and fuming nitricacid/acetic anhydride. Suitable solvents for the reaction includetrifluoroacetic acid. The reaction temperature is not particularlylimited and is usually −20° C. to room temperature, and preferablereaction temperatures include 0-10° C.

Step A-(xi):

This step is a step of obtaining a compound A-(12) byt-butoxycarbonylation of the amino group of the compound A-(11).

The reaction can be performed under the same conditions as thosegenerally used in t-butoxycarbonylation of an amino compound such as theconditions described in a document such as T. W. Greene and P. G. M.Wuts, “Protective Groups in Organic Chemistry, Third Edition”, JohnWiley & Sons (1999), P. 518-525. The compound A-(12) can be obtained byreacting the compound A-(11) with di-tert-butyl dicarbonate using in asolvent such as tetrahydrofuran, for example. Alternative solventsinclude acetonitrile and DMF. Those skilled in the art will appreciatethat a base may also be added to the reaction mixture, although is notessential. Suitable examples of a base include, but are not limited totriethylamine and diisopropylethylamine. The reaction temperature is notparticularly limited and is usually to room temperature to reflux, andpreferably room temperature to 60° C.

Step A-(xii):

This step is a step of obtaining a compound A-(13) from the compoundA-(12).

The compound A-(13) is synthesized by reducing the nitro compound A-(12)by a synthesis method known to a person skilled in the art. Examples ofthe method include reduction by catalytic hydrogenation using a noblemetal catalyst such as Raney nickel, palladium, ruthenium, rhodium orplatinum. Other reducing reagents include tin chloride, for example.Examples of the solvent include alcoholic solvents such as methanol,ethanol and 1-propanol, preferably ethanol. The reaction time is notparticularly limited and is usually 0.5 to 24 hours, and preferably 0.5to 18 hours. The reaction temperature is usually room temperature.Alternative reduction reaction conditions include reaction with ironwith an additive such as ammonium chloride or hydrochloric acid, in analcoholic solvent such as ethanol, at an appropriate reactiontemperature, for example 65° C.

Step A-(xiii):

This is a step of obtaining a compound A-(14) from the compound A-(13)by condensing compound A-(13) with a carboxylic acid and a condensingagent. The condensation reaction can be performed under the sameconditions as those usually used and described in the followingdocuments. Examples of the known method include those in Rosowsky etal.; J. Med. Chem., 34 (1), 227-234 (1991), Brzostwska et al.;Heterocycles, 32 (10), 1968-1972 (1991), and Romero et al.; J. Med.Chem., 37 (7), 998-1014 (1994).

The compound A-(13) may be a free form or a salt.

The solvent in this reaction is not particularly limited insofar as itdoes not inhibit the reaction. Examples of the solvent includetetrahydrofuran, 1,4-dioxane, ethyl acetate, methyl acetate,dichloromethane, chloroform, N,N-dimethylformamide, toluene,acetonitrile and xylene. Examples of the condensing agent include CDI(N,N′-carbonyldiimidazole), Bop(1H-1,2,3-benzotriazol-1-yloxy(tri(dimethylamino))phosphoniumhexafluorophosphate), WSC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride), DCC(N,N-dicyclohexylcarbodiimide), diethylphosphorylcyanide, PyBOP (benzotriazol-1-yloxytri(pyrrolidino)phosphoniumhexafluorophosphate) and EDC.HCl(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride). Suitableconditions include an agent to activate the acid, such as N,N′-carbonyldiimidazole. One equivalent to a large excess of the acid may be usedwith respect to the compound A-(13). One equivalent to a large excess ofan organic base such as triethylamine or N,N-diisopropylethylamine maybe added where necessary.

The reaction time is not particularly limited and is usually 0.5 to 72hours, and preferably 0.5 to 24 hours. The reaction temperature variesaccording to the raw material used, the solvent and the like and is notparticularly limited. Ice-cold temperature to solvent reflux temperatureis acceptable, ice cold to room temperature is preferable.

Alternatively, the compound A-(14) can be obtained by converting thedesired carboxylic acid to the corresponding acid chloride and thenreacting the acid chloride with the compound A-(13). The acid chloridecan be synthesized by a means known to a person skilled in the art. Forexample the desired carboxylic acid may converted to the correspondingacid chloride by reaction with thionyl chloride in the presence orabsence of a solvent, for example dichloromethane,N,N′-dimethylimidazoline-2-one, NMP or DMF. One to two equivalents or alarge excess of thionyl chloride may be used with respect to the desiredcarboxylic acid. The reaction temperature is −30° C. to reflux, andpreferably −10° C. to room temperature. The acid chloride may also beformed by treating the acid with oxalyl chloride in a solvent such asdichloromethane in the presence of DMF. The reaction temperature is −30°C. to room temperature, and preferably −10° C. to room temperature

Alternatively, the compound A-(14) can be obtained by converting thedesired carboxylic acid to a mixed acid anhydride and then reacting themixed acid anhydride with the compound A-(13). The mixed acid anhydridecan be synthesized by a means known to a person skilled in the art. Thesynthesis is performed by reacting the desired carboxylic acid with achloroformate such as ethyl chloroformate in the presence of a base suchas triethylamine, for example. One to two equivalents of thechloroformate and the base are used with respect to the desiredcarboxylic acid. The reaction temperature is −30° C. to roomtemperature, and preferably −20° C. to room temperature.

The step of condensing the mixed acid anhydride with the compound 1-(13)is performed by reacting the mixed acid anhydride with the compound1-(13) in a solvent such as dichloromethane, tetrahydrofuran orN,N-dimethylformamide, for example. One equivalent to a large excess ofthe desired carboxylic acid is used with respect to the compound A-(13).

The reaction time is not particularly limited and is usually 0.5 to 48hours, and preferably 0.5 to 12 hours. The reaction temperature is −20°C. to 50° C., and preferably −20° C. to room temperature.

Alternatively, the compound A-(14) can be obtained by converting thedesired carboxylic acid to an active ester and then reacting the activeester with the compound A-(13). The step of obtaining the active esteris performed by reacting the desired carboxylic acid with an activeester synthesis reagent in a solvent such as 1,4-dioxane,tetrahydrofuran or N,N-dimethylformamide in the presence of a condensingagent such as DCC, for example. Examples of the active ester synthesisreagent include N-hydroxysuccinimide. One to 1.5 equivalents of theactive ester synthesis reagent and the condensing agent are used withrespect to the compound A-(13). The reaction time is not particularlylimited and is usually 0.5 to 48 hours, and preferably 0.5 to 24 hours.

The reaction temperature is −20° C. to 50° C., and preferably −20° C. toroom temperature.

The step of condensing the active ester with the compound A-(13) isperformed by reacting the active ester with the compound A-(13) in asolvent such as dichloromethane, tetrahydrofuran orN,N-dimethylformamide, for example. One equivalent to a large excess ofthe active ester is used with respect to the compound A-(13). Thereaction time is not particularly limited and is usually 0.5 to 48hours, and preferably 0.5 to 24 hours. The reaction temperature is −20°C. to 50° C., and preferably −20° C. to room temperature.

Step A-(xiv):

This step is a step of obtaining the compound A-(15) by deprotection ofthe t-butoxycarbonyl group of the compound A-(14).

The reaction can be performed under the same conditions as thosegenerally used in deprotection reaction of a t-butoxycarbonyl group suchas the conditions described in a document such as T. W. Greene and P. G.M. Wuts, “Protective Groups in Organic Chemistry, Third Edition”, JohnWiley & Sons (1999), P. 518-525. The compound A-(15) can be obtained byreacting the compound I-(14) with a strong acid, for exampletrifluoroacetic acid in the presence or absence of a solvent. Suitablesolvents include dichloromethane. Alternative acids include hydrochloricacid in suitable solvents, such as dichloromethane or dioxane, forexample.

The reaction temperature is normally ice cold to 80° C., preferably roomtemperature. The reaction time is not particularly limited and isusually 5 minutes to 48 hours, and preferably 5 minutes to 12 hours.

B. General Preparation Method B:

In the formula, X, Y and A are as defined above.

General Preparation Method B is an alternative method for preparing acompound A-(15) which corresponds to compound (I) according to thepresent invention from a compound A-(11) as a raw material throughmultiple steps of Step B-(i) to Step B-(ii).

The compound A-(11) may be prepared as described in General PreparationMethod A or the examples.

Step B-(i):

This step is a step of obtaining a compound A-(16) from the compoundA-(11).

The compound A-(16) is synthesized by reducing the nitro compound A-(11)by a synthesis method known to a person skilled in the art. Examples ofthe method include reduction by catalytic hydrogenation using a noblemetal catalyst such as Raney nickel, palladium, ruthenium, rhodium orplatinum. Other reducing reagents include tin chloride, for example.Examples of the solvent include alcoholic solvents such as methanol,ethanol and 1-propanol, preferably ethanol. The reaction time is notparticularly limited and is usually 0.5 to 24 hours, and preferably 0.5to 18 hours. The reaction temperature is usually room temperature.Alternative reduction reaction conditions include reaction with ironwith an additive such as ammonium chloride or hydrochloric acid, in analcoholic solvent such as ethanol, at an appropriate reactiontemperature, for example 65° C.

Step B-(ii):

This is a step of obtaining a compound A-(15) from the compound A-(16)by condensing compound A-(13) with a carboxylic acid and a condensingagent. The condensation reaction can be performed under the sameconditions as those usually used and described in the followingdocuments. Examples of the known method include those in Rosowsky etal.; J. Med. Chem., 34 (1), 227-234 (1991), Brzostwska et al.;Heterocycles, 32 (10), 1968-1972 (1991), and Romero et al.; J. Med.Chem., 37 (7), 998-1014 (1994).

The compound A-(15) can be obtained by converting the desired carboxylicacid to the corresponding acid chloride and then reacting the acidchloride with the compound A-(16). The acid chloride can be synthesizedby a means known to a person skilled in the art. For example the desiredcarboxylic acid may converted to the corresponding acid chloride byreaction with thionyl chloride in the presence or absence of a solvent,for example dichloromethane, N,N′-dimethylimidazoline-2-one, NMP or DMF.One to two equivalents or a large excess of thionyl chloride may be usedwith respect to the desired carboxylic acid. Those skilled in the artwill appreciate that the choice of reaction conditions employed mayaffect the outcome of the reaction, for example the conditions mayaffect whether the acid chloride reacts with the aniline or theisothiourea moieties. Those skilled in the art will appreciate that thereaction of thionyl chloride with a carboxylic acid results in theconcomitant formation of 1 equivalent of hydrochloric acid in additionto the formation of the desired acid chloride. Those skilled in the artwill appreciate that the current conditions do not employ a method ofremoving the thus formed hydrochloric acid. The hydrochloric acid formedin this reaction may or may not affect the selectivity of the reactionwhich may or may not result in a beneficial outcome. The reaction timeis not particularly limited and is usually 0.5 to 48 hours, andpreferably 0.5 to 12 hours. The reaction temperature is −30° C. toreflux, and preferably −10° C. to room temperature. The acid chloridemay also be formed by treating the acid with oxalyl chloride in asolvent such as dichloromethane in the presence of DMF. The reactiontemperature is −30° C. to room temperature, and preferably −10° C. toroom temperature.

C. General Preparation Method C:

General Preparation Method C is an alternative method for preparing acompound A-(2) which is a synthetic intermediate of the compound (I)according to the present invention from a compound A-(17) as a rawmaterial through Step C-(i).

The compound A-(17) is commercially available.

Step i:

This step is a step of obtaining a compound A-(2a) from A-(17) by addinga trifluoromethyl anion to the compound A-(17) to generate anintermediate allylic alkoxide or intermediate trimethylsilyl ether whichis then alkylated to give the compound A-(2a). Those skilled in the artwill appreciate that this transformation can be conducted in one pot oras two individual reactions. Those skilled in the art will appreciatethe benefits and drawbacks of a one pot reaction compared to conductingtwo separate reactions and choose the best method for their requirementsaccordingly.

Specifically, acrolein A-(17) can react with a trifluoromethyl anionwhich can be generated by the action of fluoride on reagents such as(trifluoromethyl)trimethylsilane to generate the corresponding allylicalkoxide or allylic timethylsilyl ether. The solvent used in thereaction is not particularly limited insofar as it does not interferewith the reaction. Examples of suitable solvents include THF. Acceptabletemperature ranges for the reaction include −10° C. to solvent reflux,preferably below room temperature. Those skilled in the art willappreciate that certain chemical reactions can be exothermic and thatcontrol measures should be put in place to control these exotherms.Those skilled in the art will also appreciate that the reaction exothermmay be controlled by allowing the solvent to reflux. Suitable precursorsto generate trifluoromethyl anion include, but are not limited to,(trifluoromethyl)trimethylsilane (Rupert's reagent, Chem Rev 1997, 97,757) and suitable fluoride sources include, but are not limited to,tetrabutylammonium fluoride (TBAF), tetrabutylammoniumdifluorotriphenylsilicate (TBAT) and caesium fluoride. Although theinitial reaction temperature may be below room temperature it isacceptable to allow the reaction temperature to reach solvent refluxduring the course of the reaction to facilitate reaction. The reactiontime is not particularly limited and is usually 5 minutes to 24 hours,preferably 1-6 hours.

Those skilled in the art will appreciate that the alkoxide generatedfrom this reaction can be reacted with an alkylating agent directly,such as tert-butyl bromoacetate, and that this reaction may proceed withor without additional solvents. If additional solvents are required tofacilitate reaction, then solvents such as DMF or NMP are suitable. Thereaction temperature is not particularly limited. Suitable reactiontemperatures include room temperature to 80° C., preferably roomtemperature. The reaction time is not particularly limited and isusually 5 minutes to 1 week, preferably 1-48 hours. Alternatively, thealkylation reaction can be conducted under phase transfer conditions,for example by adding an aqueous base, for example aqueous sodiumhydroxide. Those skilled in the art will appreciate that when theseconditions are applied the use of a phase transfer catalyst is required.Suitable phase transfer catalysts include, but are not limited to,tetrabutylammonium hydrogen sulfate.

Those skilled in the art will appreciate that the intermediate alkoxidecould be quenched, isolated and purified then subjected to independentalkylation conditions. This reaction can be performed under the sameconditions as those usually used in O-alkylation reaction of an alcoholcompound (such as the conditions described in Tetrahedron Lett. 46(2005) 45, 7751-7755). In this reaction, the compound A-(2a) can beobtained by adding a base such as sodium hydride to a solution of theintermediate alcohol in THF to prepare an alkoxide, and then reactingthe alkoxide with the tert-butyl bromoacetate, for example. The solventused in the reaction is not particularly limited insofar as it does notinhibit the reaction and allows the starting material to be dissolvedtherein to a certain extent. Examples of the solvent include solventssuch as THF, DMF and dimethyl sulfoxide. The reaction can be performedby causing 1 to 3 equivalents of an appropriate base to act in thepresence of such a solvent. Examples of the base used include sodiumhydride, potassium hydride and t-butoxypotassium. The reaction time isnot particularly limited and is usually 0.5 to 72 hours, and preferably0.5 to 12 hours. The reaction temperature is usually −20° C. to 50° C.

A more preferable result such as an improved yield may be achieved byadding a salt such as tetrabutylammonium iodide in this reaction.

Those skilled in the art will appreciate that this reaction generates anew chiral centre in the compound A-(2a) and that compound A-(2a) is thesame as compound A-(2) except that compound A-(2) is enantiomericallypure whereas compound A-(2a) is racemic. It will be appreciated by thoseskilled in the art the enantiomerically pure compound A-(2) and theracemic compound A-(2a) are indistinguishable by analytical techniquessuch as NMR and liquid chromatography, however they are distinguishableby chiral HPLC. Those skilled in the art will appreciate that the mostappropriate methods to obtain the desired enantiomer from the compoundA-(2a) or a more advanced synthetic intermediate or final compound.Appropriate methods and appropriate stages of enantiomeric purificationinclude those as detailed in the examples.

In a further aspect, the present invention provides a process ofpreparing a compound of formula (I), which comprises reacting a compoundof formula A-(16), wherein X is defined in formula (I), with a compoundof formula (II) wherein A and Y are as defined in formula (I), or a C₁₋₆alkyl ester, acid anhydride or acid halide thereof, to yield a compoundof formula (I), and optionally converting the compound to a furthercompound of formula (I) or forming a pharmaceutically acceptable saltthereof

The reaction of A-(16) and (II) may conveniently be conducted in asolvent (such as tetrahydrofuran, 1,4-dioxane, ethyl acetate, methylacetate, dichloromethane, chloroform, N,N-dimethylformamide, toluene,acetonitrile or xylene) at a temperature in the range of −30° C. to 100°C. In one embodiment of the invention, compound (II) may convenientlytake the form of an acid halide (e.g. chloride) as may be prepared byreacting the acid with a suitable reagent (e.g. thionyl chloride)

It will be appreciated by those skilled in the art that in the processof the present invention certain functional groups such as hydroxyl,carboxyl or amino groups in the starting reagents may need to beprotected by protecting groups. Thus the preparation of the compounds offormula (I) may additionally involve incorporation and removal of one ormore protecting groups. The protection and deprotection of functionalgroups is for example described in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Chemistry, Third Edition”, John Wiley &Sons (1999).

The present invention will be described more specifically below withreference to Examples, Preparation Examples and Test Example. However,the present invention is not limited thereto. The abbreviations used inExamples are conventional abbreviations known to a person skilled in theart. Some abbreviations are shown below:

LCMS, LC/MS & LC-MS (liquid chromatography/mass spectrometry); MS (massspectrometry); MDAP (mass directed auto purification); NMR (nuclearmagnetic resonance); s, d, t, dd, m, br (singlet, doublet, triplet,doublet of doublets, multiplet, broad); Ph, Me, Et, Pr, Bu, Bn (phenyl,methyl, ethyl, propyl, butyl, benzyl); THF (tetrahydrofuran); DCM(dichloromethane); DMF (N,N-dimethylformamide); h, hr, hrs (hours); EDC& EDAC (N-3-(dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride);DMAP (4-N,N-dimethylaminopyridine); DMSO (dimethylsulfoxide); UV(ultraviolet); RT & rt (room temperature); Rt (retention time); min &mins (minutes); EtOAc (ethyl acetate); Et₂O (diethyl ether); MeCN(acetonitrile); EtOH (ethanol); MeOH (methanol); PhCH₃ & PhMe (toluene);tic (thin layer chromatography); TFA (trifluoroactic acid); NaOH (sodiumhydroxide); HCl (hydrochloric acid); NMP (N-methylpyrrolidinone or1-methyl-2-pyrrolidinone); HPLC (high performance liquidchromatography); TBAF (tetrabutylammonium fluoride); BuLi (n-butyllithium); PyBOP: benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate; Pd₂ dba₃: tris(dibenzylideneacetone)dipalladium;Pd(t-Bu₃P)₂: bis(tri-t-butylphosphine)palladium; TFA: trifluoroaceticacid; pTLC: preparative thin-layer chromatography; HRMS (high resolutionmass spectrometry); Tr or Trt (trityl or triphenylmethyl).

¹H NMR spectra were recorded on a Bruker AM series spectrometeroperating at a (reported) frequency of 400 MHz. Chemical shifts inproton nuclear magnetic resonance spectra are recorded in δ units (ppm)relative to tetramethylsilane and coupling constants (J) are recorded inHertz (Hz). Patterns are designated as s: singlet, d: doublet, t;triplet, br; broad.

The “room temperature” in the following Examples and PreparationExamples typically refers to about 10° C. to about 35° C. “%” indicateswt % unless otherwise specified.

Chemical names were generated from chemical structures using ChemBioDrawUltra 11.0 and 12.0.

DESCRIPTION OF FIGURES

FIG. 1 is a Typical Chromatogram from a Chiral HPLC Isolation ofCompound 1-(20).

Preparation Example 1 Synthesis of tert-butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate1-(13) 1-(2) Synthesis of tert-butyl{[(2S)-1,1,1-trifluorobut-3-en-2-yl]oxy}acetate

To a suspension of trimethylsulfonium iodide (110 g) in THF (500 mL) at−30° C. was added lithium hexamethyldisilazide (530 mL, 1N in THF)portionwise over 45 mins. After stirring at −20° C. for 20 mins,(S)-2-trifluoromethyloxirane (37.97 g) was added at the same temperatureover 15 mins, and the mixture was allowed to warm to RT and stirred for3 h. The slurry was then added portionwise to an ice-cold solution often-butyl bromoacetate (105.68 g) in NMP (200 mL). The resulting mixturewas allowed to warm to RT and stir for 2 days, before dilution withEtOAc (1 L). The organic layer was washed with sodium bicarbonate (sat.,aq., 4×400 mL), dried over MgSO₄ and evaporated. The residue waspurified by silica gel column chromatography (5% EtOAc in hexanes) toobtain the title compound (70.1 g) which was used in the subsequent stepwithout purification. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.30 (s, 9 H)3.83-3.96 (m, 2H) 4.14-4.21 (m, 1H) 5.34-5.48 (m, 2H) 5.56-5.71 (m, 1H)

1-(3) Synthesis of(S)—N-methoxy-N-methyl-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)acetamide

tert-Butyl {[(2S)-1,1,1-trifluorobut-3-en-2-yl]oxy}acetate (70.1 g,crude) was dissolved in ice-cold formic acid (200 mL). The mixture wasallowed to warm to RT and stir overnight. The reaction mixture was thenconcentrated under reduced pressure, toluene (200 mL) was added, themixture concentrated, before a second addition of toluene (200 mL) andconcentration to an oil. The residue was dissolved in DCM (600 mL),cooled in an ice-bath, and N,N′-carbonyl diimidazole (35 g) was addedportionwise over 20 mins. After stirring for 45 mins, N,O-dimethylhydroxylamine hydrochloride (22 g) was added, and the reaction mixturewas allowed to warm to RT and stir overnight. Saturated NaHCO₃ (500 mL)and brine (250 mL) were then added, and the mixture extracted with EtOAc(3×750 mL). The combined organic portions were dried over MgSO₄ andevaporated. The residue was purified by silica gel column chromatography(1% to 30% EtOAc in hexanes) to obtain the title compound (25.17 g).¹H-NMR (400 MHz, CDCl₃) δ (ppm) 3.21 (s, 3H), 3.71 (m, 3H), 4.36-4.51(m, 3H), 5.54-5.69 (m, 2H), 5.84 (ddd, J=17.7, 10.4, 7.3 Hz, 1H)

1-(4) Synthesis of(S)-1-(2-fluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanone

A solution of n-butyllithium in hexane (2.50 M; 90 mL) was addeddropwise over 25 mins to a solution of 2-bromofluorobenzene (40.35 g) inTHF (250 mL) under a N₂ atmosphere at −78° C. The reaction solution wasallowed to warm to −60° C. and stir for 60 min.(S)—N-Methoxy-N-methyl-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)acetamide(40 g) in THF (25 mL) was added dropwise to the reaction solution, andafter stirring at −60° C. for 2 h, aqueous NH₄Cl (100 mL) was added tothe reaction solution, followed by warming to RT. Brine (200 mL) wasadded to the reaction solution, and the mixture was extracted with EtOAc(3×400 mL). The combined organic portions were dried over MgSO₄,evaporated, and the residue was purified by silica gel columnchromatography (1% to 10% EtOAc in hexanes) to obtain the title compound(33.59 g). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 4.40 (pentet, J=6.3 Hz, 1H)4.81-4.87 (m, 2H), 5.54-5.69 (m, 2H), 5.86 (ddd, J 17.4, 10.4, 7.3 Hz,1H) 7.12-7.22 (m, 1H) 7.24-7.34 (m, 1H) 7.54-7.63 (m, 1H) 7.94-8.02 (m,1H).

1-(5) Synthesis of(S)-1-(2-fluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanoneoxime

(S)-1-(2-Fluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanone(41.22 g) was dissolved in anhydrous methanol (400 mL) and hydroxylaminehydrochloride (14.0 g) and sodium acetate (19.0 g) were added. Thereaction mixture was heated to 50° C. for 90 min, then cooled to RT,concentrated in vacuo and the residue purified by silica gelchromatography (2% to 15% EtOAc in hexanes) to afford the title compoundas a mixture of geometric isomers (40.54 g). ¹H-NMR (400 MHz, CDCl₃) δ(ppm): 4.04-4.15 (m, 0.8H), 4.18-4.26 (m, 0.2H), 4.44-4.57 (m, 0.4H)4.79-4.90 (m, 1.6H) 5.37-5.56 (m, 2H) 5.64-5.78 (m, 1H) 7.03-7.26 (m,2H) 7.33-7.54 (m, 2H), 7.90 (br s, 0.2H), 8.51 (br s, 0.8H).

1-(6) Synthesis of(3aR,4S,6a5)-6a-(2-fluorophenyl)-4-(trifluoromethyl)hexahydrofuro[3,4-c]isoxazole

(S)-1-(2-fluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanoneoxime (40.54 g) was dissolved in xylenes (400 mL) and hydroquinone (4.0g) was added. The reaction mixture was heated to reflux (heating blocktemperature 140° C.) for 22 h, then cooled and evaporated. The residuewas purified by silica gel column chromatography (1% to 30% EtOAc inhexanes) to obtain the title compound (28.76 g). ¹H-NMR (400 MHz, CDCl₃)δ (ppm): 3.71-3.81 (m, 1H), 4.04-4.35 (m, 4H), 4.51-4.62 (m, 1H),5.38-5.54 (m, 1H), 7.07-7.26 (m, 2H), 7.32-7.42 (m, 1H), 7.54-7.67 (m,1H).

1-(7)((2S,3R,4S)-4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol

(3aR,4S,6aS)-6a-(2-fluorophenyl)-4-(trifluoromethyl)hexahydrofuro[3,4-c]isoxazole(28.76 g) was dissolved in acetic acid (200 mL) and the solution wascooled to 0° C. Zinc (50 g) was added, and the reaction mixture wasallowed to warm and stir at RT for 16 h. The reaction mixture was thendiluted with EtOAc (500 mL) and filtered through celite, washing with afurther 500 mL of EtOAc. The combined organic portions were evaporated,dissolved in chloroform (200 mL), and ammonia (28% aq., 250 mL) wasadded slowly. The layers were separated, and the aqueous portion wasfurther extracted with chloroform (2×250 mL). The combined organicextracts were dried over anhydrous MgSO₄ and evaporated to afford thetitle compound (31.12 g) which was used in the subsequent step withoutfurther purification. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 2.93 (ddd, J=7.7,4.9, 2.5 Hz, 1H), 3.84 (dd, J=12.4, 4.8 Hz, 1H), 4.05 (dd, J=9.2, 3.2Hz, 1H), 4.17 (dd, J=12.4, 2.3 Hz, 1H), 4.31 (d, J=9.3 Hz, 1H), 4.72(quin, J=7.3 Hz, 1H), 7.13 (ddd, J=13.1, 8.8, 1.3 Hz, 1H), 7.22 (td,J=7.6, 1.3 Hz, 1H), 7.31-7.40 (m, 1H), 7.51 (td, J=8.0, 1.6 Hz, 1H)

1-(8) Synthesis ofN-(((3S,4R,5S)-3-(2-fluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)tetrahydrofuran-3-yl)carbamothioyl)benzamide

Benzoyl isothiocyanate (19.0 mL) was added to a solution containing((2S,3R,45)-4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol(28.72 g) in DCM (150 mL), and the mixture was stirred at RT for 18 h.Sodium bicarbonate (sat., aq., 200 mL) was then added, the mixtureextracted with EtOAc (3×300 mL), dried over MgSO₄ and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (5% to 30% EtOAc in hexanes) to obtain the title compound(37.07 g). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 3.22 (dd, J=8.1, 4.5 Hz,1H), 3.31 (td, J=8.0, 3.0 Hz, 1H), 3.94-4.07 (m, 1H), 4.31-4.46 (m, 1H),4.53 (d, J=9.9 Hz, 1H), 4.83 (d, J=9.9 Hz, 1H), 6.97-7.14 (m, 1H), 7.22(td, J=7.7, 1.3 Hz, 1H), 7.31-7.45 (m, 1H), 7.49-7.61 (m, 2H), 7.61-7.70(m, 1H), 7.75 (td, J=8.1, 1.5 Hz, 1H), 7.79-7.93 (m, 2H), 8.90 (s, 1H),11.85 (s, 1H)

1-(9) Synthesis ofN-((4aS,5S,7aS)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide

N-(((3S,4R,5S)-3-(2-Fluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)tetrahydrofuran-3-yl)carbamothioyl)benzamide(31.1 g) was dissolved in pyridine (150 mL), and the mixture cooled to−20° C. Trifluoromethanesulfonic anhydride (14.0 mL) was added dropwiseover 30 min and the reaction was allowed to warm to 0° C. After stirringfor 2 h, the reaction was quenched by the addition of ammonium chloride(sat., aq., 400 mL) and extracted with EtOAc (3×500 mL). The combinedorganic extracts were dried over MgSO₄, concentrated in vacuo andpurified by silica gel column chromatography (2% to 30% EtOAc/hex) toobtain the title compound (18.50 g). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.86(dd, J=13.9, 3.5 Hz, 1H), 3.25 (d, J=13.6 Hz, 1H), 3.61 (br. s., 1H),4.00-4.10 (m, 1H), 4.66 (d, J=8.8 Hz, 1H), 4.78-4.87 (m, 1H), 7.12-7.60(m, 6H), 7.68-7.73 (m, 1H), 7.99-8.16 (br. s., 2H), 8.62-8.66 (m, 1H)

1-(10) Synthesis ofN-((4aS,5S,7a5)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]-thiazin-2-yl)benzamide

N-((4aS,5S,7aS)-7a-(2-Fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide(21.5 g) was dissolved in methanol (160 mL),1,8-diazabicyclo[5.4.0]undec-7-ene (16.29 g) was added, and the solutionwas heated to reflux (heating block temperature 80° C.). After 16 h, thereaction mixture was concentrated under reduced pressure, and theresidue purified by silica gel column chromatography (10% to 60% EtOAcin hexanes) to afford the title compound (13.82 g). ¹H NMR (400 MHz,CDCl₃) δ ppm 2.85 (dd, J=13.6, 3.8 Hz, 1H), 3.14 (dd, J=13.5, 3.2 Hz,1H), 3.33-3.45 (m, 1H), 3.92 (dd, J=8.1, 2.0 Hz, 1H), 4.49 (br. s., 2H),4.63-4.76 (m, 2H), 7.08 (ddd, J=12.6, 8.1, 1.0 Hz, 1H), 7.13-7.22 (m,1H), 7.25-7.36 (m, 1H), 7.44 (td, J=8.0, 1.9 Hz, 1H)

1-(11) Synthesis of(4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

N-((4aS,5S,7aS)-7a-(2-Fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide(5.15 g) was dissolved in TFA (75 mL), and the solution was cooled to 0°C. Sulfuric acid (conc., 20 mL) was added, followed by fuming nitricacid (2 mL) dropwise over 20 mins. After stirring at 0° C. for 90 mins,the reaction mixture was poured onto ice (200 g) and basified to pH 12with 6N NaOH (aq.). After allowing the ice to melt, the mixture wasextracted with EtOAc (3×500 mL), and the combined organic portions driedover MgSO₄ and evaporated to afford the title compound (22.1 g, purityapprox. 71%) which was used in the subsequent step without purification.¹H NMR (400 MHz, CDCl₃) δ ppm 2.89 (d, J=3.8 Hz, 1H), 3.09 (br. s., 1H), 3.28-3.54 (m, 1H), 3.80-4.03 (m, 1H), 4.50-4.70 (m, 3H), 4.71-4.86(m, 1H), 7.21-7.30 (m, 1H), 8.18-8.28 (m, 1H), 8.45 (dd, J=6.8, 2.8 Hz,1H)

1-(12) Synthesis of tert-butyl((4aS,5S,7a5)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate

(4aS,5S,7aS)-7a-(2-Fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(20.6 g, crude) was dissolved in THF (300 mL), di-tert-butyl dicarbonate(12 g) was added portionwise over 20 mins and the reaction mixture washeated to 60° C. Further portions of di-tert-butyl dicarbonate (10 g)were added until starting material was consumed by TLC. The reactionmixture was cooled and sodium bicarbonate (sat., aq., 200 mL) and brine(200 mL) were added. The mixture was then extracted with EtOAc (3×500mL) and the combined organic portions were dried over MgSO₄ andevaporated. The residue was purified by silica gel column chromatography(10% to 25% EtOAc in hexanes) to afford the title compound (16.62 g). ¹HNMR (400 MHz, CDCl₃) δ ppm 1.55 (s, 9H), 2.73-2.84 (m, 1H), 2.92-3.05(m, 1H), 3.43-3.55 (m, 1H), 3.81-3.94 (m, 1H), 4.57 (d, J=8.3 Hz, 1H),4.73-4.83 (m, 1H), 7.19-7.39 (m, 2H), 8.20-8.29 (m, 1H), 8.32 (d, J=6.8Hz, 1H)

1-(13) Synthesis of tert-butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate

tert-Butyl((4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate(16.61 g) was dissolved in ethanol (250 mL) and tin chloride dihydrate(25.0 g) was added. After stirring at RT for 18 h, the solution waspoured onto NaOH (2N aq., 300 mL) and Celite® (˜50 g) was added. Theresulting mixture was filtered through more Celite® and extracted withEtOAc (2×500 mL). The combined organic portions were dried over MgSO₄and evaporated to afford the title compound (15.52 g). This materialcould be used crude but a portion was purified by silica gel columnchromatography (20% to 50% EtOAc in hexanes) to afford pure material(recovery 79%). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.53 (s, 9H), 2.77 (d,J=14.4 Hz, 1H), 3.09 (br. s., 1H), 3.46 (br. s., 1H), 3.62 (br. s., 2H),3.87 (br. s., 1H), 4.61 (d, J=8.6 Hz, 1H), 4.71 (br. s., 1H), 6.61 (br.s., 2H), 6.85-6.95 (m, 1H)

Preparation Example 2 Synthesis of tert-butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate2-(10)

2-(1) Synthesis of(S)-1-(2,3-difluorophenyl)-2((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanone

A solution of n-butyllithium in hexane (2.50 M, 13.5 mL) was addeddropwise over 20 mins to a solution containing1-bromo-2,3-difluorobenzene (6.50 g) in Et₂O (50 mL) under a N₂atmosphere at −78° C. The reaction solution was allowed to stir for 60mins.(S)—N-Methoxy-N-methyl-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)acetamide)(5.20 g) in Et₂O (10 mL) was then added dropwise to the reactionsolution, and after stirring at −78° C. for 1 h, aqueous NH₄Cl (50 mL)was added to the reaction solution, followed by warming to RT. NaHCO₃(sat. aq., 100 mL) was added to the reaction solution, and the mixturewas extracted with EtOAc (3×100 mL). The combined organic portions weredried over MgSO₄, evaporated, and the residue was purified by silica gelcolumn chromatography (1% to 10% EtOAc in hexanes) to obtain the titlecompound (3.91 g). ¹H NMR (400 MHz, CDCl₃) δ ppm: 4.33-4.43 (m, 1H),4.80-4.84 (m, 2H), 5.55-5.67 (m, 2H), 5.76-5.94 (m, 1H), 7.18-7.28 (m,1H), 7.37-7.47 (m, 1H), 7.70 (ddt, J=7.9, 6.0, 1.7 Hz, 1H)

2-(2) Synthesis of(S)-1-(2,3-difluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanoneoxime

(S)-1-(2,3-difluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanone(3.91 g) was dissolved in anhydrous methanol (40 mL) and hydroxylaminehydrochloride (1.25 g) and sodium acetate (1.68 g) were added. Thereaction mixture was heated to 50° C. for 90 min, then cooled to RT,concentrated in vacuo and the residue purified by silica gelchromatography (2% to 20% EtOAc in hexanes) to afford the title compoundas a mixture of geometric isomers (4.10 g). ¹H-NMR (400 MHz, CDCl₃) δ(ppm): 4.04-4.26 (m 1H), 4.43-4.55 (m, 0.4H) 4.80-4.89 (m, 1.6H)5.39-5.55 (m, 2H) 5.64-5.80 (m, 1H) 7.05-7.30 (m, 3H), 7.76 (br s,0.2H), 8.30 (br s, 0.8H).

2-(3) Synthesis of(4S)-6a-(2,3-difluorophenyl)-4-(trifluoromethyl)hexahydrofurol-3,4-c]isoxazole

(S)-1-(2,3-difluorophenyl)-2-((1,1,1-trifluorobut-3-en-2-yl)oxy)ethanoneoxime (4.10 g) was dissolved in xylenes (40 mL) and hydroquinone (380mg) was added. The reaction mixture was heated to reflux (heating blocktemperature 140° C.) for 20 h, then cooled and evaporated. The residuewas purified by silica gel column chromatography (1% to 25% EtOAc inhexanes) to obtain the title compound (3.16 g). ¹H NMR (400 MHz, CDCl₃)δ ppm 3.77 (br. s., 1H), 3.99-4.16 (m, 1H), 4.16-4.22 (m, 1H), 4.22-4.44(m, 2H), 4.51 (d, J=9.9 Hz, 1H), 5.44 (s, 1H), 7.07-7.24 (m, 2H), 7.38(br. s., 1H)

2-(4) Synthesis of((2S,3R,4S)-4-amino-4-(2,3-difluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol

(4S)-6a-(2,3-difluorophenyl)-4-(trifluoromethyl)hexahydrofuro[3,4-c]isoxazole(3.16 g) was dissolved in acetic acid (20 mL) and the reaction mixturecooled to 0° C. Zinc (5.0 g) was added, and the reaction was allowed towarm and stir at RT for 20 h. The reaction mixture was then diluted withEtOAc (50 mL) and filtered through Celite®, washing with a further 100mL of EtOAc. The combined organic portions were evaporated, dissolved inCHCl₃ (20 mL), and ammonia (28% aq., 25 mL) was added slowly. The layerswere separated, and the aqueous portion was further extracted with CHCl₃(2×25 mL). The combined organic extracts were dried over anhydrous MgSO₄and evaporated to afford the title compound (3.12 g) which was used inthe subsequent step without further purification. ¹H NMR (400 MHz,CDCl₃) δ ppm 2.93 (ddd, J=7.8, 5.1, 2.8 Hz, 1H), 3.85 (dd, J=12.4, 5.1Hz, 1H), 4.03 (dd, J=9.1, 2.8 Hz, 1H), 4.14 (dd, J=12.3, 2.7 Hz, 1H),4.35 (d, J=9.1 Hz, 1H), 4.68 (quin, J=7.3 Hz, 1H), 7.09-7.25 (m, 2H),7.25-7.34 (m, 1H)

2-(5) Synthesis ofN-(((3S,4R,5S)-3-(2,3-difluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)tetrahydrofuran-3-yl)carbamothioyl)benzamide

Benzoyl isothiocyanate (2.0 mL) was added to a solution containing((2S,3R,4S)-4-amino-4-(2,3-difluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol(3.12 g) in DCM (20 mL), and the mixture was stirred at RT for 18 h.Sodium bicarbonate (sat., aq., 50 mL) was then added, the mixtureextracted with EtOAc (3×75 mL), dried over MgSO₄ and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (5% to 40% EtOAc in hexanes) to obtain the title compound(4.18 g). ¹H NMR (400 MHz, CDCl₃) δ ppm 3.12 (dd, J=7.6, 4.3 Hz, 1H),3.18-3.29 (m, 1H), 4.03 (ddd, J=12.3, 7.2, 4.5 Hz, 1H), 4.35-4.49 (m,1H), 4.59 (d, J=9.9 Hz, 1H), 4.81 (d, J=9.6 Hz, 1H), 7.07-7.23 (m, 2H),7.49 (t, J=7.2 Hz, 1H), 7.56 (t, J=7.7 Hz, 2H), 7.67 (t, J=7.5 Hz, 1H),7.88 (d, J=7.1 Hz, 2H), 8.92 (s, 1H), 11.89 (s, 1H)

2-(6) Synthesis ofN-((4aS,5S,7aS)-7a-(2,3-difluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide

N-(((3S,4R,5S)-3-(2,3-Difluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)tetrahydrofuran-3-yl)carbamothioyl)benzamide(2.99 g) was dissolved in pyridine (14 mL), and the mixture cooled to−20° C. Trifluoromethanesulfonic anhydride (1.55 mL) was added dropwiseover 10 min and the reaction mixture was allowed to warm to −10° C.After stirring for 2 h, a further portion of trifluoromethanesulfonicanhydride (1.0 mL) was added dropwise over 10 min, the reaction wasstirred for a further 2 h, and was then quenched by the addition ofammonium chloride (sat., aq., 50 mL) and extracted with EtOAc (3×100mL). The combined organic extracts were dried over MgSO₄, concentratedin vacuo and purified by silica gel column chromatography (5% to 20%EtOAc/hex) to obtain the title compound (1.20 g). ¹H NMR (400 MHz,CDCl₃) δ ppm 2.86 (d, J=10.6 Hz, 1H), 3.20 (br. s., 1H), 3.55 (br. s.,1H), 4.04 (br. s., 1H), 4.65 (d, J=8.8 Hz, 1H), 4.81 (br. s., 1H),7.06-7.24 (m, 3H), 7.40-7.64 (m, 3H), 7.82-8.21 (m, 2H)

2-(7) Synthesis of(4aS,5S,7aS)-7a-(2,3-difluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

N-((4aS,5S,7aS)-7a-(2,3-Difluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide(2.00 g) was dissolved in methanol (250 mL),1,8-diazabicyclo[5.4.0]undec-7-ene (1.53 g) was added, and the solutionwas heated to reflux (heating block temperature 80° C.). After 3 h, thereaction mixture was concentrated under reduced pressure, diluted withwater (100 mL) and extracted with EtOAc (3×100 mL). The combined organicportions were dried over MgSO₄, evaporated, and the residue purified bysilica gel column chromatography (0% to 50% EtOAc in hexanes) to affordthe title compound (1.42 g). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.86 (dd,J=13.6, 3.8 Hz, 1H), 3.15 (dd, J=13.8, 3.2 Hz, 1H), 3.27-3.42 (m, 1 H),3.93 (dd, J=8.2, 1.9 Hz, 1H), 4.39-4.78 (m, 4H), 6.96-7.25 (m, 3H)

2-(8) Synthesis of(4aS,5S,7aS)-7a-(2,3-difluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

(4aS,5S,7aS)-7a-(2,3-Difluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(1.42 g) was dissolved in TFA (6 mL), and the solution was cooled to 0°C. Sulfuric acid (conc., 1 mL) was added, followed by fuming nitric acid(0.30 mL) dropwise over 20 mins. After stirring at 0° C. for 1 h, thereaction mixture was poured onto ice (50 g) and basified to pH 12 with 2N NaOH (aq.). After allowing the ice to melt, the mixture was extractedwith EtOAc (3×75 mL), and the combined organic portions dried over MgSO₄and evaporated to afford the title compound (1.91 g, purity approx. 80%)which was used in the subsequent step without purification. ¹H NMR (400MHz, CDCl₃) δ ppm 2.88 (dd, J=13.8, 3.9 Hz, 1H), 3.11 (dd, J=13.6, 2.8Hz, 1H), 3.37 (dt, J=7.4, 3.5 Hz, 1H), 3.93 (d, J=7.8 Hz, 1H), 4.53-4.83(m, 4H), 8.09 (ddd, J=9.0, 6.3, 2.9 Hz, 1H), 8.27 (dt, J=5.2, 2.6 Hz,1H)

2-(9) Synthesis of tert-butyl((4aS,5S,7aS)-7a-(2,3-difluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate

(4aS,5S,7aS)-7a-(2,3-Difluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(1.91 g, crude) was dissolved in THF (10 mL), di-tert-butyl dicarbonate(1.3 g) was added portionwise over 20 mins and the reaction mixture washeated to 65° C. After 3 h, the reaction mixture was cooled and sodiumbicarbonate (sat., aq., 50 mL) was added. The mixture was then extractedwith EtOAc (3×750 mL) and the combined organic portions were dried overMgSO₄ and evaporated. The residue was purified by silica gel columnchromatography (0% to 20% EtOAc in hexanes) to afford the title compound(1.43 g crude, in a mixture with the bis-boc version).

2-(10) Synthesis of tert-butyl((4aS,5S,7a5)-7a-(5-amino-2,3-difluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate

The crude mixture obtained in preparation example 2-(9) containingtert-Butyl((4aS,5S,7aS)-7a-(2,3-difluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate)(1.43 g) was dissolved in ethanol (25 mL) and tin chloride dihydrate(2.50 g) was added. After stirring for 18 h, the solution was pouredonto NaOH (2N aq., 100 mL) and extracted with EtOAc (3×100 mL). Thecombined organic portions were dried over MgSO₄, evaporated and purifiedby silica gel column chromatography (0% to 30% EtOAc in hexanes) toafford firstly the bis-boc product (730 mg), and secondly the titlecompound (300 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.53 (s, 9H), 2.76 (dd,J=13.9, 3.8 Hz, 1H), 3.08 (d, J=13.6 Hz, 1H), 3.36-3.45 (m, 1H), 3.71(br. s., 2H), 3.90 (d, J=8.3 Hz, 1H), 4.57 (d, J=8.6 Hz, 1H), 4.68-4.79(m, 1H), 6.30-6.37 (m, 1H), 6.42-6.49 (m, 1H)

Preparation Example 3 Synthesis of 5-ethoxypyrazine-2-carboxylic acid(3-(2)

Synthesis of ethyl 5-ethoxypyrazine-2-carboxylate 3-(1)

A stirred solution of methyl 5-chloropyrazine-2-carboxylate (0.50 g) inethanol (10 mL) was cooled to 0° C., and sodium ethoxide (21% w/wsolution in ethanol, 1 mL) was added over 10 mins. After allowing towarm to RT and stir for 2 h, water (100 mL) was added and the mixtureextracted with EtOAc (2×150 mL). The combined organic portions weredried over MgSO₄ and evaporated to afford the title compound. (0.65 g,purity approx. 85%). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.45 (t, J=7.1 Hz, 3H), 1.46 (t, J=7.1 Hz, 3H), 4.48 (q, J=7.1 Hz, 2H), 4.49 (q, J=7.1 Hz,2H), 8.28 (d, J=1.3 Hz, 1H), 8.88 (d, J=1.3 Hz, 1H)

Synthesis of 5-ethoxypyrazine-2-carboxylic acid 3-(2)

Ethyl 5-ethoxypyrazine-2-carboxylate (0.65 g, approx. purity 85%) wasdissolved in dioxan (3 mL) and water (3 mL) was added, followed bylithium hydroxide monohydrate (255 mg, portionwise over 10 mins). Afterstirring at RT for 24 h, Et₂O (25 mL) and NaHCO₃ (sat., aq., 25 mL) wereadded. The layers were separated and the organic layer was extractedwith NaOH (1 N, aq., 25 mL). The combined aqueous portions wereacidified with 6N HCl to pH 2, and the mixture extracted with EtOAc(3×40 mL). The combined EtOAc extracts were dried over MgSO₄ andevaporated to afford the title compound as an off-white powder. ¹H NMR(400 MHz, CDCl₃) δ ppm 1.47 (t, J=7.1 Hz, 3H), 4.53 (q, J=7.1 Hz, 2H),8.16 (d, J=1.2 Hz, 1H), 8.98 (d, J=1.2 Hz, 1H)

Preparation Example 4 Synthesis of 5-ethoxypyrazine-2-carboxylic acid(4-(3))

Methyl 5-acetylpyrazine-2-carboxylate 4-(1)

5-Acetylpyrazine-2-carboxamide (3.275 g) was dissolved in methanolic HCl(1.25 N, 150 mL) and the reaction mixture was heated to reflux andstirred overnight. After cooling, sodium bicarbonate was added and themixture was extracted with EtOAc.

The EtOAc layer was dried over MgSO₄ and evaporated to afford the titlecompound (3.79 g, approx purity 90%). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.78(s, 3H), 4.10 (s, 3H), 9.33 (d, J=1.5 Hz, 1H), 9.36 (d, J=1.5 Hz, 1H)

Methyl 5-(1,1-difluoroethyl)pyrazine-2-carboxylate 4-(2)

Methyl 5-acetylpyrazine-2-carboxylate (300 mg, approx purity 90%) wasdissolved in DCM (15 mL) and cooled to 0° C. under nitrogen.Bis(2-methoxyethyl)aminosulfur trifluoride (0.61 mL) was added dropwiseand the reaction mixture allowed to warm to RT and stir overnight.Sodium bicarbonate (sat., aq.) was added cautiously and the mixtureextracted with DCM. The organic portions were dried over MgSO₄,evaporated and purified by silica gel chromatography (35% EtOAc inhexane) to afford the title compound (155 mg) as a white solid. ¹H NMR(400 MHz, CDCl₃) δ ppm 2.00 (t, J=18.8 Hz, 3H), 4.01 (s, 3H), 8.98 (d,J=1.5 Hz, 1H), 9.24 (d, J=1.5 Hz, 1H)

5-(1,1-Difluoroethyl)pyrazine-2-carboxylic acid 4-(3)

Methyl 5-(1,1-difluoroethyl)pyrazine-2-carboxylate (0.65 g, approx.purity 85%) was dissolved in dioxan (2 mL) and water (2 mL) was added,followed by lithium hydroxide monohydrate (54 mg, portionwise). Afterstirring at RT for 90 mins, the mixture was concentrated to 2 mL andEt₂O (20 mL) added. The mixture was then extracted with NaOH (1 N, aq.,20 mL), and the aqueous portions acidified with 6N HCl to pH 2. Theaqueous portion was then extracted with EtOAc, dried over MgSO₄ andevaporated to afford the title compound as a white solid (119 mg). ¹HNMR (400 MHz, CDCl₃) δ ppm 2.11 (t, J=18.8 Hz, 3H), 9.01 (d, J=1.3 Hz,1H), 9.47 (d, J=1.3 Hz, 1H)

Preparation Example 5 Synthesis of 5-(fluoromethyl)pyrazine-2-carboxylicacid (5-(3))

Methyl 5-(hydroxymethyl)pyrazine-2-carboxylate 5-(1)

To a solution of methyl 5-formylpyrazine-2-carboxylate (2.47 g) in THF(20 mL) was added sodium borohydride (170 mg) portionwise over 10 mins.After stirring for 1 h, methanol (10 mL) was added. The reaction mixturewas stirred for a further 20 mins, and then HCl (1 N, aq., 20 mL) andbrine (20 mL) were added. The mixture was extracted with EtOAc (3×40 mL)and the combined organic portions dried over MgSO₄ and evaporated toafford the title compound (1.31 g). ¹H NMR (400 MHz, CDCl₃) δ ppm 4.07(s, 3H), 4.98 (br. s., 2H), 8.80 (s, 1H), 9.27 (s, 1H)

Methyl 5-(fluoromethyl)pyrazine-2-carboxylate 5-(2)

To a solution of methyl 5-(hydroxymethyl)pyrazine-2-carboxylate (0.64 g)in THF (20 mL) was added triethylamine (2.30 g) and the solution wascooled to 0° C. Triethylamine trihydrofluoride (1.22 g) was then addedfollowed by nonafluorobutanesulfonyl fluoride (2.28 g) dropwise over 5mins. After warming to RT and stirring for 2 h, NaHCO₃ (sat., aq., 100mL) was added, and the mixture was extracted with EtOAc (2×50 mL). Thecombined organic portions were dried over MgSO₄, evaporated, andpurified by silica gel chromatography (5% to 50% EtOAc in hexane) toafford the title compound (94 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 4.07(s, 3H), 5.67 (d, J=46.5 Hz, 2H), 8.89 (s, 1H), 9.28 (s, 1H)

5-(Fluoromethyl)pyrazine-2-carboxylic acid 5-(3)

Methyl 5-(fluoromethyl)pyrazine-2-carboxylate (94 mg) was dissolved indioxan (1 mL) and water (1 mL) was added, followed by lithium hydroxidemonohydrate (60 mg). After stirring at RT for 18 h, Et₂O (20 mL) wasadded and the mixture was then extracted with NaOH (1 N, aq., 2×20 mL).The aqueous portions were acidified with 6N HCl to pH 1, extracted withEtOAc (2×40 mL), the combined organic portions dried over MgSO₄ andevaporated to afford the title compound as a white solid (71 mg). ¹H NMR(400 MHz, CDCl₃) δ ppm 5.70 (d, J=46.2 Hz, 2H), 8.85 (s, 1H), 9.40 (s,1H)

Preparation Example 6 Synthesis of 5-difluoromethylpyrazine-2-carboxylicacid (6-(5)

Synthesis of t-butyl 5-methylpyrazine-2-carboxylate 6-(1)

A boron trifluoride-diethyl ether complex (91.7 μL) was added dropwiseto a suspension of 2-methylpyrazine-5-carboxylic acid (1 g) andtert-butyl 2,2,2-trichloroacetimidate (4.75 g) in THF (20 mL) underice-cooling. The reaction solution was warmed to RT, followed bystirring for 2 h. A saturated NaCl solution and EtOAc were added to thereaction solution, and the organic layer was separated. The organiclayer was dried over anhydrous MgSO₄, and the insoluble matter wasseparated by filtration. The filtrate was concentrated and purified bysilica gel column chromatography to obtain the title compound (1.4 g).¹H-NMR (CDCl₃) δ (ppm): 1.65 (s, 9H), 2.65 (s, 3H), 8.57 (d, J=1.2 Hz,1H), 9.10 (d, J=1.6 Hz, 1H).

Synthesis of t-butyl5-((E)-2-dimethylamino-vinyl)-pyrazine-2-carboxylate 6-(2)

A mixture of t-butyl 5-methylpyrazine-2-carboxylate (1.35 g), DMF (25mL) and N,N-dimethylformamide dimethylacetal (25 mL) was stirred at 130°C. for 5 h. The reaction solution was cooled to RT and diluted withEtOAc. The mixture was washed with a saturated NaCl solution threetimes. The organic layer was dried over anhydrous MgSO₄, and theinsoluble matter was separated by filtration. The filtrate wasconcentrated and the residue was purified by silica gel columnchromatography to obtain the title compound (648 mg). ¹H-NMR (CDCl₃) δ(ppm): 1.63 (s, 9H), 3.00 (s, 6H), 5.16 (d, J=12.8 Hz, 1H), 7.72 (d,J=12.8 Hz, 1H), 8.16 (d, J=1.2 Hz, 1H), 8.81 (d, J=1.6 Hz, 1H).

Synthesis of t-butyl 5-formylpyrazine-2-carboxylate 6-(3)

Sodium periodate (1.67 g) was added to a solution of t-butyl5-((E)-2-dimethylamino-vinyl)-pyrazine-2-carboxylate (645 mg) in 50%THF-water (26 mL), and the mixture was stirred at RT for 4 h. Asaturated NaHCO₃ solution and EtOAc were added to the reaction solution,and the organic layer was separated. The organic layer was washed with asaturated NaCl solution and dried over anhydrous MgSO₄. The insolublematter was separated by filtration and the filtrate was concentrated.The residue was purified by silica gel column chromatography to obtainthe title compound (249 mg). ¹H-NMR (CDCl₃) δ (ppm): 1.68 (s, 9H), 9.25(d, J=1.2 Hz, 1H), 9.36 (d, J=1.6 Hz, 1H), 10.2 (s, 1H).

Synthesis of t-butyl 5-difluoromethylpyrazine-2-carboxylate 6-(4)

[Bis(2-methoxyethyl)amino]sulfur trifluoride (662 μL) was added dropwiseto a solution of t-butyl 5-formylpyrazine-2-carboxylate (249 mg) inCH₂Cl₂ (12 mL) under a N₂ atmosphere under ice-cooling. The reactionsolution was stirred for 2 h while gradually returning to RT. Asaturated NaHCO₃ solution and EtOAc were added to the reaction solution,and the organic layer was separated. The organic layer was washed with asaturated NaCl solution and dried over anhydrous MgSO₄. The insolublematter was separated by filtration and the filtrate was concentrated.The residue was purified by silica gel column chromatography to obtainthe title compound (175 mg). ¹H-NMR (CDCl₃) δ (ppm): 1.67 (s, 9H), 6.75(t, J=54.4 Hz, 1H), 9.02 (d, J=0.8 Hz, 1H), 9.25 (d, J=0.8 Hz, 1H).

Synthesis of 5-difluoromethylpyrazine-2-carboxylic acid 6-(5)

Trifluoroacetic acid (1 mL) was added to a solution of t-butyl5-difluoromethylpyrazine-2-carboxylate (175 mg) in dichloromethane (1mL), and the mixture was stirred at RT for 5 h. Ether and 5 N NaOH wereadded to the reaction solution. The aqueous layer was separated and madeacidic with 5 N hydrochloric acid. EtOAc was added to the aqueous layer,and the organic layer was separated. The organic layer was dried overanhydrous MgSO₄, and the insoluble matter was separated by filtration.The filtrate was concentrated to obtain the title compound (100 mg).¹H-NMR (CDCl₃) δ (ppm): 6.80 (t, J=54.4 Hz, 1H), 9.02 (s, 1H), 9.47 (s,1H).

Preparation Example 7 Synthesis of 5-cyanopyridine-2-carboxylic acid(7-(2))

Synthesis of methyl 5-cyanopyridine-2-carboxylate 7 (1)

A mixture of methyl 5-bromopyridine-2-carboxylate (2.8 g) and coppercyanide (3.6 g) in NMP (30 mL) was heated with stirring at 170° C. for1.5 h. Water was added to the reaction solution at RT, and the insolublematter was removed by filtration. The filtrate was extracted with EtOAc.The extract was washed with a saturated NaCl solution and then driedover anhydrous MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The resulting crudeproduct was purified by silica gel column chromatography (EtOAc-heptanesystem) to obtain the title compound (920 mg). ¹H-NMR (400 MHz, CDCl₃) δ(ppm): 4.06 (s, 3H), 8.16 (dd, J=2.0, 8.0 Hz, 1H), 8.27 (d, J=8.0 Hz,1H), 9.01 (d, J=2.0 Hz, 1H).

Synthesis of 5-cyanopyridine-2-carboxylic acid 7-(2)

A solution of the compound of Preparation Example 13-(1) (920 mg) and a5 N NaOH solution (2.26 mL) in ethanol (30 mL) was stirred at RT for 10min. 5 N hydrochloric acid (5.2 mL) was added to the reaction solutionat RT, followed by extraction with EtOAc. The extract was dried overanhydrous MgSO₄. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure to obtain the titlecompound (800 mg). ¹H-NMR (400 MHz, DMSOd₆) δ (ppm): 8.18 (d, J=8.0 Hz,1H), 8.51 (dd, J=2.0, 8.0 Hz, 1H), 9.12-9.18 (m, 1H).

Preparation Example 8 Synthesis of5-(methoxymethyl)pyrazine-2-carboxylic acid (8-(2))

Synthesis of methyl 5-(methoxymethyl)pyrazine-2-carboxylate 8-(1)

The compound obtained in preparation example 5-(1) (279 mg) wasdissolved in DMF and the solution was cooled to 0° C. Sodium hydride(60% in mineral oil, 70 mg) was added, followed by iodomethane (250 mg).After 2 days, water (25 mL) was added, and the solution extracted withEtOAc (100 mL). The aqueous layer was saturated with NaCl, and furtherextracted with EtOAc (2×50 mL). The combined organic portions were driedover MgSO₄, evaporated, and purified by silica gel chromatography (30%to 50% EtOAc in hexanes) to afford the title compound (55 mg, approx.purity 65%). ¹H NMR (400 MHz, CDCl₃) δ ppm 3.55 (s, 3H), 4.05 (s, 3H),4.72 (s, 2H), 8.84 (d, J=1.0 Hz, 1H), 9.25 (d, J=1.0 Hz, 1H)

Synthesis of 5-(methoxymethyl)pyrazine-2-carboxylic acid 8-(2)

Methyl 5-(methoxymethyl)pyrazine-2-carboxylate, 8-(1), (55 mg, crude)was dissolved in 1,4-dioxane (1 mL) and water (1 mL) was added followedby lithium hydroxide monohydrate (50 mg). After stirring at RT for 1 h,water (20 mL) was added and the mixture was extracted with ether (20mL). The aqueous portion was acidified to pH 2 and extracted with EtOAc(2×25 mL). The combined EtOAc layers were dried over MgSO₄ andevaporated to afford the title compound (19 mg). ¹H NMR (400 MHz, CDCl₃)δ ppm 3.58 (s, 3H), 4.77 (s, 2H), 8.80 (br. s., 1H), 9.38 (br. s., 1H)

Preparation Example 9 5-Methoxypyrazine-2-carboxylic acid

5-Chloropyrazine-2-carboxylic acid (5.0 g, 0.032 mol) was charged to around-bottom flask equipped with a thermocouple, overhead stirrer andreflux condenser. Methanol (37.5 mL, 0.926 mol) was charged followed byconc. sulfuric acid (0.2 mL, 0.004 mol). The 3-neck flask was equippedwith a heating mantle, and then the reaction mixture was heated to ca.65.0° C. (T internal). The reaction mixture continued to stir at ca.65.0° C. (T internal) for ca. 4 h. The reaction mixture cooled to ca.25.8° C. (T internal). Methanol (12 mL, 0.31 mol) was charged and theslurry continued to stir at ca. 22.3° C. (T internal) for ca. 15 minthen cooled to ca. 10.0° C. (T internal) under an atmosphere ofnitrogen. 25% Sodium methoxide in methanol (1:3, Sodiummethoxide:Methanol, 7.7 mL) was charged to flask while temperatureremained below 30.0° C. (T internal). The reaction mixture was adjustedto 20.4° C. (T internal). After 30 min., sodium hydroxide (2.0 g, 0.04mol) and water (37.5 mL, 2.08 mol) were combined to form a solution, andthen the solution was charged to the reaction mixture. Water (50.0 mL,2.78 mol) was charged and then the reaction mixture was heated to 40.0°C. (T internal) for ca. 60 mins. The heating mantle was removed, andthen the reaction mixture cooled to ca. 25.4° C. (T internal). 38% aq.HCl Solution (38:62, hydrogen chloride:water, 4.0 mL) was added at arate (ca. 5 min.) such that the temperature remained below 30.0° C. (Tinternal). The thick slurry was stirred for 1 h at ca. 21.4° C. (Tinternal), and then filtered over a sintered funnel. The solids wererinsed with water (10.0 mL, 0.555 mol) and dried under vacuum overnightto afford 5-methoxypyrazine-2-carboxylic acid (3.59 g). ¹H NMR (500 MHz,DMSO) δ 13.24 (1H, br s), 8.79 (1H, d, J=1.2 Hz), 8.37 (1H, d, J=1.2Hz), 3.98 (s, 3H); ¹³C NMR (125 MHz, DMSO) δ 165.36, 161.88, 143.88,136.82, 135.55, 54.69.

General procedure for the Coupling of Anilines Prepared in PreparationExamples 1-(13) and 2-(10) with aryl carboxylic Acids: Preparation ofN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide(Example 1)

tert-Butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate(Preparation Example 1, 56 mg) was dissolved in DCM (2 mL) and5-methoxypyrazine-2-carboxylic acid (40 mg), N,N-diisopropylethylamine(80 mg) and (1H-benzotriazol-1-yloxy)tripyrrolidin-1-yl)phosphoniumhexafluorophosphate (135 mg) were added. The reaction mixture wasstirred at RT for 18 h, and sodium bicarbonate (sat., aq., 25 mL) wasadded. The mixture was extracted with EtOAc (2×40 mL), the combinedorganic portions were dried over MgSO₄, evaporated and purified bysilica gel chromatography (EtOAc/hexanes gradient) to afford the amide(40 mg) as a white solid. The amide was dissolved in DCM (2 mL) and TFA(1 mL) was added. After stirring at RT for 2 h, the reaction mixture wasevaporated and sodium bicarbonate (sat., aq., 25 mL) was added. Themixture was extracted with EtOAc (2×25 mL), and the combined organicportions dried over MgSO₄ and evaporated to afford the title compound asa white solid (34 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.78-2.87 (m, 1H),3.11-3.21 (m, 1H), 3.38-3.48 (m, 1H), 3.82-4.06 (m, 4H), 4.48-4.72 (m,2H), 6.96-7.10 (m, 1H), 7.52 (d, J=4.8 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H),8.09 (s, 1H), 8.95 (s, 1H), 9.46 (br. s., 1H)

Example 2N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-cyanopicolinamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-cyanopyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.82 (dd, J=13.6, 3.5 Hz, 1H),3.14-3.21 (m, 1H), 3.37-3.45 (m, 1H), 3.91 (d, J=9.1 Hz, 1H), 4.50-4.71(m, 2H), 7.08 (dd, J=11.9, 8.8 Hz, 1H), 7.46-7.57 (m, 1H), 7.92 (dt,J=8.8, 3.4 Hz, 1H), 8.17 (dd, J=8.3, 2.0 Hz, 1H), 8.34 (d, J=8.1 Hz,1H), 8.81-8.92 (m, 1H)

Example 3N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-difluoromethyl-pyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.80 (dd, J=13.8, 3.7 Hz, 1H),3.11 (dd, J=13.6, 2.8 Hz, 1H), 3.31-3.41 (m, 1H), 3.86 (d, J=8.3 Hz,1H), 4.57 (d, J=8.3 Hz, 1H), 4.64 (dt, J=14.7, 7.1 Hz, 1H), 4.75 (br s,2H), 6.69 (t, J=56.3 Hz, 1H), 7.07 (dd, J=11.6, 8.8 Hz, 1H), 7.57 (dd,J=7.1, 2.8 Hz, 1H), 7.87 (dt, J=8.5, 3.6 Hz, 1H), 8.85 (s, 1H), 9.45 (s,1H), 9.58 (br. s., 1H)

Example 4N-(3-((4aS,5S,7a5)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(trifluoromethyl)picolinamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-trifluoromethyl-pyridine-2-carboxylic acid according to thegeneral procedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.88 (dd, J=13.6, 3.8Hz, 1H), 3.20 (dd, J=13.6, 3.0 Hz, 1H), 3.37-3.53 (m, 1H), 3.94 (dd,J=8.3, 2.3 Hz, 1H), 4.40-4.89 (m, 4H), 7.14 (dd, J=11.9, 8.8 Hz, 1H),7.66 (dd, J=6.8, 2.8 Hz, 1H), 7.98 (ddd, J=8.8, 4.0, 3.0 Hz, 1H), 8.20(dd, J=8.2, 1.6 Hz, 1H), 8.45 (d, J=8.3 Hz, 1H), 8.90 (s, 1H), 9.95 (s,1H)

Example 5N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methylpyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-methyl-pyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.72 (s, 3H), 2.88 (dd, J=13.6,3.8 Hz, 1H), 3.21 (dd, J=13.6, 3.0 Hz, 1H), 3.42-3.49 (m, 1H), 3.94 (d,J=8.3 Hz, 1H), 4.39-4.80 (m, 4H), 7.14 (dd, J=11.9, 8.8 Hz, 1H), 7.62(dd, J=7.1, 2.8 Hz, 1H), 7.93-7.99 (m, 1H), 8.46 (d, J=1.0 Hz, 1H), 9.39(d, J=1.3 Hz, 1H), 9.65 (s, 1H)

Example 6N-(3-((4aS,5S,7a5)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methylpicolinamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-methyl-pyridine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.37 (s, 3H), 2.78 (dd, J=13.6,3.8 Hz, 1H), 3.12 (dd, J=13.6, 2.8 Hz, 1H), 3.28-3.40 (m, 1H), 3.87 (d,J=8.1 Hz, 1H), 4.28-5.02 (m, 4H), 7.02 (dd, J=11.9, 8.8 Hz, 1H), 7.55(dd, J=7.1, 2.8 Hz, 1H), 7.63 (dd, J=8.0, 1.4 Hz, 1H), 7.88 (ddd, J=8.8,4.0, 2.9 Hz, 1H), 8.10 (d, J=8.1 Hz, 1H), 8.31-8.40 (m, 1H), 9.90 (s,1H)

Example 7N-(3-((4aS,5S,7a5)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethylpicolinamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-ethyl-pyridine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.33 (t, J=7.6 Hz, 3H), 2.78(q, J=7.6 Hz, 2H), 2.88 (dd, J=13.5, 3.7 Hz, 1H), 3.22 (dd, J=13.6, 2.8Hz, 1H), 3.42-3.48 (m, 1H), 3.96 (d, J=7.3 Hz, 1H), 4.44-4.95 (m, 4H),7.12 (dd, J=11.6, 8.8 Hz, 1H), 7.62 (dd, J=6.9, 2.7 Hz, 1H), 7.74 (dd,J=8.1, 1.8 Hz, 1H), 7.98 (dt, J=8.7, 3.5 Hz, 1H), 8.21 (d, J=8.1 Hz,1H), 8.44-8.48 (m, 1H), 10.00 (s, 1H)

Example 8N-(3-((4aS,5S,7a5)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-fluoromethyl-pyrazine-2-carboxylic acid according to the generalprocedure. Details of an actual preparation are as follows:-tert-Butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate(500 mg) was dissolved in DCM (10 mL) and5-fluoromethyl-pyrazine-2-carboxylic acid (223 mg),N,N-diisopropylethylamine (521 mg) and(1H-benzotriazol-1-yloxy)tripyrrolidin-1-yl)phosphoniumhexafluorophophate (750 mg) were added. The reaction mixture was stirredat RT for 1 h, and sodium bicarbonate (sat., aq., 50 mL) was added. Themixture was extracted with EtOAc (2×75 mL), the combined organicportions were dried over MgSO₄, evaporated and purified by silica gelchromatography (0% to 30% EtOAc/hexanes gradient) to afford the amide(613 mg) as a white solid. The amide was dissolved in DCM (2 mL) and TFA(1 mL) was added. After stirring at RT for 2 h, the reaction mixture wasevaporated and sodium bicarbonate (sat., aq., 25 mL) was added. Themixture was extracted with EtOAc (3×25 mL), and the combined organicportions dried over MgSO₄ and evaporated to afford the title compound asa white solid

¹H NMR (400 MHz, CDCl₃) δ ppm 2.89 (dd, J=13.8, 3.7 Hz, 1H), 3.21 (dd,J=13.9, 2.5 Hz, 1H), 3.40-3.51 (m, 1H), 3.96 (d, J=7.3 Hz, 1H),4.42-4.85 (m, 4H), 5.69 (d, J=46.5 Hz, 2H), 7.15 (dd, J=11.9, 8.8 Hz,1H), 7.64 (dd, J=7.1, 2.8 Hz, 1H), 7.94-8.00 (m, 1H), 8.77 (s, 1H), 9.47(s, 1H), 9.68 (s, 1H)

Example 9N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypicolinamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-methoxypyridine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.88 (dd, J=13.8, 3.7 Hz, 1H),3.23 (dd, J=13.8, 2.4 Hz, 1H), 3.46 (d, J=7.3 Hz, 1H), 3.89-4.02 (m,4H), 4.54-5.00 (m, 4H), 7.11 (dd, J=11.9, 8.8 Hz, 1H), 7.36 (dd, J=8.8,2.8 Hz, 1H), 7.61 (dd, J=7.1, 2.8 Hz, 1H), 7.97 (dt, J=8.5, 3.6 Hz, 1H),8.23-8.30 (m, 2H), 9.86 (s, 1H)

Example 10N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-ethoxypyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-ethoxypyrimidine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.47 (t, J=7.1 Hz, 3H), 2.87(dd, J=13.6, 3.5 Hz, 1H), 3.20 (d, J=13.6 Hz, 1H), 3.40-3.50 (m, 1H),3.94 (d, J=7.8 Hz, 1H), 4.46-4.95 (m, 6H), 7.11 (dd, J=11.5, 9.0 Hz,1H), 7.54-7.63 (m, 1H), 7.90-8.01 (m, 1H), 8.13 (s, 1H), 9.00 (s, 1H),9.52 (br. s., 1H)

Example 11N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(1,1-difluoroethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-(1,1-difluoroethyl)pyrazine-2-carboxylic acid according to thegeneral procedure. ¹H NMR (600 MHz, CDCl₃) δ ppm 2.03 (t, J=18.8 Hz,3H), 2.82 (d, J=12.0 Hz, 1H), 3.14 (d, J=12.4 Hz, 1H), 3.35-3.46 (m,1H), 3.77-4.07 (m, 1H), 4.20-4.93 (m, 4H), 7.08 (dd, J=11.7, 9.0 Hz,1H), 7.56 (d, J=4.5 Hz, 1H), 7.80-7.93 (m, 1H), 8.87 (s, 1 H), 9.43 (s,1H), 9.59 (br. s., 1H)

Example 12N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(trifluoromethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-trifluoromethylpyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.80 (dd, J=13.6, 3.8 Hz, 1H),3.11 (dd, J=13.8, 2.9 Hz, 1H), 3.30-3.44 (m, 1H), 3.87 (d, J=8.3 Hz,1H), 4.25-5.14 (m, 4H), 7.07 (dd, J=11.9, 8.8 Hz, 1H), 7.57 (dd, J=6.8,2.8 Hz, 1H), 7.86 (dt, J=8.4, 3.6 Hz, 1H), 8.89 (s, 1H), 9.53 (s, 2H)

Example 13N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(methoxymethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-(methoxymethyl)pyrimidine-2-carboxylic acid according to thegeneral procedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.79 (dd, J=13.6, 3.8Hz, 1H), 3.11 (dd, J=13.6, 2.8 Hz, 1H), 3.32-3.39 (m, 1H), 3.49 (s, 3H),3.84 (d, J=8.3 Hz, 1H), 4.34-4.73 (m, 6 H), 7.05 (dd, J=11.6, 8.8 Hz,1H), 7.55 (dd, J=6.8, 2.8 Hz, 1H), 7.88 (dt, J=8.4, 3.6 Hz, 1H), 8.63(s, 1H), 9.34 (d, J=1.0 Hz, 1H), 9.60 (s, 1H)

Example 14N-{3-[(4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5-dihydro-4H-furo[3,4d][1,3]thiazin-7a(7H)-yl]-4-fluorophenyl}-5-[(²H₃)methyloxy]pyrazine-2-carboxamideSynthesis of 14-(2) 5-[(²H₃)methyloxy]pyrazine-2-carboxylic acid

Part (I): (²H₃)methyl 5-[(²H₃)methyloxy]pyrazine-2-carboxylate

Freshly cut sodium metal (160 mg) was added portionwise over 10 mins to(²H₃)methan(²H)ol (5 mL) and the solution was stirred until the sodiumhad dissolved. This solution was then added to methyl5-chloropyrazine-2-carboxylate (1.02 g) in (²H₃)methan(²H)ol (5 mL) andthe solution was allowed to stir at RT for 1 hr. The solution was thenconcentrated under reduced pressure to a volume of about 2 mL, and water(50 mL) was added. The mixture was extracted with EtOAc (2×50 mL), thecombined organic portions were dried over MgSO₄ and evaporated to affordthe title compound (745 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.30 (d,J=1.3 Hz, 1H), 8.91 (d, J=1.3 Hz, 1H)

Part (II): 5-[(²H₃)methyloxy]pyrazine-2-carboxylic acid

To a stirred solution of (²H₃)methyl5-[(²H₃)methyloxy]pyrazine-2-carboxylate in 1,4-dioxane (5 mL) was addedwater (5 mL) followed by lithium hydroxide monohydrate (300 mg). Afterstirring for 1 hr, the reaction mixture was concentrated under reducedpressure to about 5 mL and extracted with diethyl ether (25 mL). Theorganic layer was extracted with 1N NaOH (aq., 10 mL), and the combinedaqueous portions were acidified to pH 2 with 6N hydrochloric acid. Aftercooling in a fridge, the mixture was filtered to afford the titlecompound as a pale brown powder (660 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm8.21 (d, J=1.3 Hz, 1H), 9.01 (d, J=1.3 Hz, 1H), 10.12 (br s., 1H)

Part (III):N-{3-[(4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5-dihydro-4H-furo[3,4d][1,3]thiazin-7a(7H)-yl]-4-fluorophenyl}-5-[(²H₃)methyloxy]pyrazine-2-carboxamide

tert-Butyl((4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)carbamate(100 mg) was dissolved in DCM (2 mL) and5-[(²H₃)methyloxy]pyrazine-2-carboxylic acid (55 mg),N,N-diisopropylethylamine (112 mg) and(1H-benzotriazol-1-yloxy)tripyrrolidin-1-yl)phosphoniumhexafluorophosphate (180 mg) were added. The reaction mixture wasstirred at RT for 18 h, and sodium bicarbonate (sat., aq., 25 mL) wasadded. The mixture was extracted with EtOAc (2×40 mL), the combinedorganic portions were dried over MgSO₄, evaporated and purified bysilica gel chromatography (2% to 25% EtOAc in hexanes) to afford theamide (127 mg) as a white solid. The amide was dissolved in DCM (2 mL)and TFA (1 mL) was added. After stirring at RT for 2 h, the reactionmixture was evaporated and sodium bicarbonate (sat., aq., 25 mL) wasadded. The mixture was extracted with EtOAc (2×40 mL), and the combinedorganic portions dried over MgSO₄ and evaporated to afford the titlecompound as a white solid (104 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 2.87(dd, J=13.6, 3.8 Hz, 1H), 3.21 (dd, J=13.6, 2.8 Hz, 1H), 3.39-3.53 (m,1H), 3.95 (d, J=8.3 Hz, 1H), 4.65 (d, J=8.3 Hz, 1H), 4.72 (quin, J=7.2Hz, 1H), 4.87 (br s, 2H), 7.12 (dd, J=11.9, 8.8 Hz, 1H), 7.60 (dd,J=6.9, 2.7 Hz, 1H), 7.95 (dt, J=8.5, 3.6 Hz, 1H), 8.16 (d, J=1.0 Hz,1H), 9.02 (d, J=1.0 Hz, 1H), 9.52 (s, 1H)

Example 15N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2,3-difluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-difluoromethyl-pyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.90 (dd, J=13.8, 3.7 Hz, 1H),3.19 (dd, J=13.8, 2.7 Hz, 1H), 3.31-3.49 (m, 1H), 3.95 (d, J=7.6 Hz,1H), 4.44-5.15 (m, 4H), 6.81 (t, J=55.8 Hz, 4H), 7.22-7.35 (m, 1H), 8.08(ddd, J=11.2, 6.8, 2.7 Hz, 1H), 8.94 (s, 1H), 9.53 (s, 1H), 9.67 (s, 1H)

Example 16N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-methoxypyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2,3-difluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-methoxypyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃+MeOD) δ ppm 2.83 (dd, J=13.9, 3.8 Hz,1H), 3.14 (dd, J=13.9, 3.0 Hz, 1H), 3.29-3.39 (m, 1H), 3.87 (d, J=8.3Hz, 1H), 4.04 (s, 3H), 4.60 (d, J=8.3 Hz, 1H), 4.67 (quin, J=6.3 Hz,1H), 7.11-7.21 (m, 1H), 8.03 (ddd, J=11.6, 6.9, 2.7 Hz, 1H), 8.15 (d,J=1.3 Hz, 1H), 8.96 (d, J=1.3 Hz, 1H)

Example 17N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-methylpyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2,3-difluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-methylpyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃+MeOD) δ ppm 2.68 (s, 3H), 2.84 (dd,J=13.6, 3.8 Hz, 1H), 3.15 (dd, J=13.9, 3.0 Hz, 1H), 3.30-3.42 (m, 1H),3.88 (d, J=10.4 Hz, 1H), 4.61 (d, J=8.6 Hz, 1H), 4.68 (quin, J=7.2 Hz,1H), 7.13-7.25 (m, 1H), 8.05 (ddd, J=11.6, 6.8, 2.8 Hz, 1H), 8.46 (s,1H), 9.30 (d, J=1.3 Hz, 1H)

Example 18N-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4,5-difluorophenyl)-5-(fluoromethyl)-pyrazine-2-carboxamide

Synthesized from tert-butyl[(4aS,5S,7aS)-7a-(5-amino-2,3-difluorophenyl)-5-trifluoromethyl-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl]carbamateand 5-(fluoromethyl)pyrazine-2-carboxylic acid according to the generalprocedure. ¹H NMR (400 MHz, CDCl₃) δ ppm 2.89 (dd, J=13.6, 3.8 Hz, 1H),3.19 (dd, J=13.6, 3.0 Hz, 1H), 3.43 (dd, J=7.5, 3.4 Hz, 1H), 3.86-3.99(m, 1H), 4.39-4.67 (m, 3H), 4.74 (quin, J=7.1 Hz, 1H), 5.76 (d, J=45.5Hz, 2H), 8.10 (ddd, J=11.4, 6.8, 2.8 Hz, 1H), 8.77 (s, 1H), 9.46 (s,1H), 9.69 (s, 1H)

Alternate Preparations of the compounds of Examples 1 and 8 aredescribed herein below. For these alternate prepartions ¹H NMR and ¹³CNMR spectra were recorded on a Varian 400 MHz or 500 MHz instrument withvNMR 6.1C software.

Alternative Preparation ofN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide(Example 1) 1-(14) Synthesis of ten-Butyl2-(1,1,1-trifluorobut-3-en-2-yloxy)acetate

A reaction vessel was charged with toluene (3.2 L), THF (0.60 L) andacrolein (0.40 L, 5.985 mol) at rt under nitrogen.(Trifluoromethyl)trimethylsilane (1.003 kg, 7.059 mol) was added at 17°C. The reaction mixture was cooled to 2.5° C. and TBAF (0.01 M in THF,0.400 L, 0.004 mol) was added over 2 h. During addition of TBAF, thetemperature of the reaction mixture increased to 65° C. The reactionmixture was cooled to 0° C., and after 2 h, tetra-n-butylammoniumhydrogen sulfate (0.171 kg, 0.503 mol) was added, followed by tert-butylbromoacetate (0.987 kg, 5.064 mol). Sodium hydroxide (50% wt in water,4.2 kg, 52.6 mol) was added over 2 h while maintaining the temperatureunder 10° C. After 2 h at 0-5° C., to the reaction mixture was addedwater (2.9 L) and methyl tert-butyl ether (6.0 L). The aq. phase wasextracted one more time with methyl tert-butyl ether (6.0 L). Theorganic phases were combined and washed with 14% aq. NaCl (3×1.6 L). Theorganics were concentrated under vacuum to afford the title compound asan oil (1.150 kg, 94.5%) which was used in the subsequent stage withoutadditional purification. ¹H NMR (500 MHz, CDCl₃) δ ppm 5.86-5.74 (m,1H), 5.59 (d, J=17.5 Hz, 1H), 5.56 (d, J=10.9 Hz, 1H), 4.37-4.30 (m,1H), 4.11 (d, J=16.5 Hz, 1H), 4.06 (d, J=16.4 Hz, 1H), 1.40 (s, 9H); ¹³CNMR (125 MHz, CDCl₃) δ ppm 168.51, 128.49 (d, J=1.7 Hz), 123.86, 123.71(q, J=281.8 Hz), 82.22, 78.67 (q, J=31.5 Hz), 66.60, 28.02.

1-(15) Synthesis ofN-Methoxy-N-methyl-2-(1,1,1-trifluorobut-3-en-2-yloxy)acetamide

To a reactor containing tert-butyl2-(1,1,1-trifluorobut-3-en-2-yloxy)acetate (1.150 kg, 4.788 mol) wasadded formic acid (6.2 kg) at rt. The reaction mixture was heated to55-60° C. for 4-5 h. The formic acid was evaporated under vacuum(T=40-45° C.) and chased with toluene (2×3.0 L). To the residue wasadded CH₂Cl₂ (2.0 L) and further concentrated under vacuum. To theresulting residue was added CH₂Cl₂ (4.6 L) and the solution was cooledto 0° C., followed by N,N-carbonyldiimidazole (1.05 kg, 6.49 mol) infive portions. The mixture was stirred for 30 mins, andN,O-dimethylhydroxylamine hydrochloride (0.67 kg, 6.72 mol) was added inportions while maintaining temperature below 10° C. The reaction mixturewas warmed to rt and stirred over 14 h. The reaction mixture was cooledto 3.2° C. and imidazole (100.7 g, 1.48 mol) was charged in twoportions. The reaction mixture was warmed to rt and water (1.4 kg) wasadded, followed by methyl tert-butyl ether (14.0 L). The organic phasewas washed with 2.0 N aq. HCl (1.0 L and 0.7 L), followed by sat. aq.NaHCO₃ (1.2 L) and sat. aq. NaCl (1.20 L). The organics wereconcentrated to afford the title compound as an oil (0.95 kg, 87.2%). ¹HNMR (500 MHz, CDCl₃) δ ppm 5.85-5.76 (m, 1H), 5.62 (d, J=17.2 Hz, 1H),5.56 (d, J=10.4 Hz, 1H), 4.49-4.34 (m, 3H), 3.68 (s, 3H), 3.67 (s, 1H),3.18 (s, 3H), 3.08 (s, 1H); ¹³C NMR (126 MHz, cdcl₃) δ ppm 169.9*,163.4*, 128.61, 123.87 (d, J=282.0 Hz), 123.82, 78.54 (q, J=31.3 Hz),66.12, 61.52, 60.56, 36.20, 32.24. Note: this compound is a 3:1 mixtureof amide bond rotamers. *Carbonyl chemical shifts estimated indirectlythrough ¹H-¹³C HMBC (heteronuclear multiple-bond correlation).

HRMS Calculated for C₈H₁₂F₃NO₃ [M+H]⁺ 228.0848; found 228.0836.

1-(16) Synthesis of1-(2-Fluorophenyl)-2-(1,1,1-trifluorobut-3-en-2-yloxy)ethanone

To a solution 1-bromo-2-fluorobenzene (0.967 kg, 5.527 mol) in THF (6.2L) at −75° C., was added n-butyllithium (2.50 M in hexane, 2.09 L, 5.22mol) while maintaining temperature below −65° C. (ca. 100 min.). After15 mins, a solution ofN-methoxy-N-methyl-2-(1,1,1-trifluorobut-3-en-2-yloxy)acetamide (0.949kg, 4.178 mol) in THF (1.6 L) was added while maintaining temperaturebelow −65° C. (ca. 70 min.). After 2.5 h at −78° C., the reaction wasquenched by addition of sat. aq. NH₄Cl (3.0 L) and methyl tert-butylether (9.0 L). The reaction mixture was warmed to rt, the aq. phase wasextracted again with methyl tert-butyl ether (2.5 L). The organic phaseswere combined, washed with sat. aq. NaCl (2×0.3 L) and concentratedunder vacuum to afford the title compound as an oil (1.007 kg, 80.0%).¹H NMR (500 MHz, CDCl₃) δ ppm 7.96 (td, J=7.6, 1.8 Hz, 1H), 7.62-7.54(m, 1H), 7.33-7.25 (m, 1H), 7.20-7.12 (m, 1H), 5.86 (ddd, J=17.5, 10.4,7.3 Hz, 1H), 5.60 (dd, J=20.5, 13.8 Hz, 2H), 4.91-4.76 (m, 2H), 4.39(dq, J=12.8, 6.4 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δ ppm 193.55, 162.14(d, J_(CF)=254.1 Hz), 135.36 (d, J_(CF)=9.2 Hz), 130.62 (d, J_(CF)=3.2Hz), 128.49, 124.85 (d, J_(CF)=3.3 Hz), 123.89, 122.93, 122.72 (d,J_(CF)=24.5 Hz), 116.50 (d, J_(CF)=23.7 Hz), 78.97 (q, J_(CF)=31.4 Hz),74.56 (d, J_(CF)=12.4 Hz).

HRMS Calculated for C₁₂H₁₀F₄O₂ [M+H]⁺ 263.0695; found 263.0709.

1-(17) Synthesis of1-(2-Fluorophenyl)-2-(1,1,1-trifluorobut-3-en-2-yloxy)ethanone oxime

To a reactor was added hydroxylamine hydrochloride (0.34 kg, 4.95 mol),sodium acetate (0.47 kg, 5.70 mol) and MeOH (2.68 L). To this suspensionwas charged a solution of1-(2-fluorophenyl)-2-(1,1,1-trifluorobut-3-en-2-yloxy)ethanone (0.998kg, 3.806 mol) in MeOH (1.8 L) and the reaction mixture was heated to40-50° C. Upon completion (ca. 2 h) the reaction mixture was cooled tort, and filtered over Celite (0.5 wt) and rinsed with EtOAc (3.0 L). Thefiltrate was concentrated under vacuum and to the resulting residue wasadded methyl tert-butyl ether (6.3 L), water (0.94 L) and sat. aq.NaHCO₃ (2.5 L). The organic phase was washed once with water (1.6 L) andsat. aq. NaCl (0.1 L). The organic phase was concentrated under vacuumto afford the title compound as an oil (1.03 kg, 95.0%). ¹H NMR (500MHz, CDCl₃) δ ppm 7.49-7.35 (m, 2H), 7.24-7.06 (m, 2H), 5.78-5.65 (m,1H), 5.54-5.40 (m, 2H), 4.89-4.81 (m, 1H), 4.53 (d, J=12.6 Hz, 1H), 4.47(d, J=12.6 Hz, 0.5H), 4.27-4.18 (m, 1H), 4.13-4.05 (m, 0.5H).

HRMS Calculated for C₁₂H₁₁F₄NO₂ [M+H]⁺ 278.0804; found 278.0780.

Note: 1-(2-Fluorophenyl)-2-(1,1,1-trifluorobut-3-en-2-yloxy)ethanoneoxime exists as an equilibrium of structural isomers, which accounts forthe less-than-whole-number integral values.

1-(18) Syntheis of(3aR*,4S*,6aS*)-6a-(2-fluorophenyl)-4-(trifluoromethyl)hexahydrofuro[3,4-c]isoxazole

To a solution of1-(2-fluorophenyl)-2-(1,1,1-trifluorobut-3-en-2-yloxy)ethanone oxime(1.085 kg, 3.328 mol) in xylenes (6.9 L) was added hydroquinone (86.2 g,0.8 mol) at rt. The solution was heated to 128° C. (internaltemperature) for 18 h. The solution was cooled to rt, and hexanes (7.0L) was added, followed by 4.0 M aq. HCl (2.4 L). The reaction mixturewas stirred for 1 h, and filtered. To the solid was added water (2.0 L),methyl tert-butyl ether (7.0 L) and 25% wt. aq. NaOH (0.4 L). The aq.layer was extracted once with methyl tert-butyl ether (7.0 L), theorganics were combined, washed with 27% aq. NaCl (2.0 L) andconcentrated under vacuum to a black oil (512.0 g, 56%). ¹H NMR (500MHz, CDCl₃) δ ppm 7.64-7.52 (m, 1H), 7.39-7.31 (m, 1H), 7.19 (td, J=7.7,1.2 Hz, 1H), 7.11 (ddd, J=11.9, 8.2, 1.0 Hz, 1H), 4.54 (d, J=10.1 Hz,1H), 4.34-4.23 (m, 1H), 4.26-4.17 (m, 1H), 4.16 (d, J=10.2 Hz, 1H), 4.10(d, J=8.5 Hz, 1H), 3.71 (d, J=20.2 Hz, 1H); ¹³C NMR (125 MHz, CDCl₃) δppm 160.59 (d, J_(CF)=247.0 Hz), 130.50 (d, J_(CF)=8.7 Hz), 128.72,124.69 (d, J_(CF)=3.3 Hz), 124.45 (q, J_(CF)=281.8 Hz), 124.43 (d,J_(CF)=11.9 Hz), 116.66 (d, J_(CF)=22.7 Hz), 83.70 (q, J_(cF)=32.1 Hz),78.17 (d, J_(CF)=3.1 Hz), 77.63. 54.53.

HRMS Calculated for C₁₂H₁₁F₄NO₂ [M+H]⁺ 278.0804; found 278.0802.

1-(19) Synthesis of)((2S*,3R*,4S*)-4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol

Zinc (389.2 g, 5.95 mol) was placed in a reaction vessel, and water wasadded (893 mL). Acetic acid (135 mL, 2.38 mol) was added whilemaintaining the temperature below 10° C. After 15 min,6a-(2-fluorophenyl)-4-(trifluoromethyl)hexahydrofuro[3,4-c]isoxazole(550.0 g, 1.98 mol) was added as a solution in THF (665 mL). Thereaction mixture was stirred over 16 h at rt. Methylene chloride (1.89L) was added, followed by 28% aq. NH₄OH (552 mL) while the temperaturewas kept below 30° C. The mixture was stirred for 30 min, and thenfiltered over Celite (80 g) rinsing with methylene chloride (378 mL).The aq. layer was extracted with methylene chloride (1.89 L). Theorganics were combined, washed with sat. aq. NaCl (1.0 L) andconcentrated under vacuum to afford an oil (502 g, 90.6%). The cruderesidue was used in the following step without additional purification.

HRMS Calculated for C₁₂H₁₃F₄NO₂ [M+H]⁺ 280.0961; found 280.0972.

1-(20) Synthesis of((2S,3R,4S)-4-Amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol(2S,3S)-2,3-bis(benzoyloxy)succinate

To a solution of4-amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol(0.502 kg, 1.798 mol) in ethanol (4.865 L) was addeddibenzoyl-D-tartaric acid (0.642 kg, 1.798 mol). The resultingsuspension was heated to 67° C. Water (94.0 mL, 5.2 mol) was added over15 min while maintaining temperature >66° C. The resulting solution wascooled to to 45° C. while precipitation occurred. The slurry wasreheated to 60° C., and then cooled to ambient temperature at 5°C./hour. The slurry was filtered, and the solid was rinsed with premixedand cooled solution of ethanol (950 mL) and water (20 mL). The solid wasdried until constant weight under vacuum (370 g, 97.6% ee). ¹H NMR (500MHz, CD₃OD) δ ppm 8.13 (d, J=7.2 Hz, 4H), 7.66-7.58 (m, 3H), 7.54-7.45(m, 5H), 7.36-7.20 (m, 2H), 5.92 (s, 2H), 4.79-4.66 (m, 1H), 4.40-4.28(m, 1H), 4.04 (dd, J=12.1, 3.4 Hz, 1H), 3.92 (dd, J=12.1, 5.4 Hz, 1H),3.30-3.24 (m, 1H); ¹³C NMR (125 MHz, DMSO) δ ppm 169.61, 165.81, 160.23(d, J=246.1 Hz), 133.00, 131.34 (d, J=9.1 Hz), 129.65, 129.55, 128.08,127.97 (d, J=3.5 Hz), 124.95 (d, J=3.3 Hz), 116.56 (d, J=23.5 Hz), 77.48(q, J_(CF)=31.0 Hz), 76.33, 73.20, 65.61 (d, J=3.1 Hz), 57.11.

HRMS Calculated for C₁₂H₁₃F₄NO₂ [M+H]⁺ 280.0961; found 280.0967 (foramino alcohol).

The absolute stereochemistry of the title compound was assigned bycomparison with a sample prepared starting from enantioenriched(S)-2-(trifluoromethyl)oxirane.

Chiral HPLC Parameters:

Equipment, Reagents, and Mobile Phase:

Equipment: HPLC column: Chiralcel OD, 4.6 × 250 mm, 10 μm, DaicelChemical Industries, Ltd., catalog no. 14025. Solvent Delivery Agilent1100 HPLC ternary pump, low pressure mixing System: with in-linedegasser, or equivalent. Autosampler: Agilent 1100 autosampler, 0.1 to100 μL range, or equivalent. Detector: Agilent 1100 variable wavelengthdetector or equivalent. Chromatographic Agilent ChemStation softwareversion A.09.03 or higher Software: for HPLC, Waters Empower 2 Build2154 or equivalent. Volumetric Class A. Glassware: Volumetric Class A.pipette: Pipettor: Calibrated Eppendorf adjustable volume, orequivalent. Balance: Analytical balance, capable of weighing ±0.1 mg.Reagents: Heptane: HPLC grade, Baker (catalog no. 9177-03) orequivalent. 2-Propanol: HPLC grade, Baker (catalog no. 9095-03) orequivalent. Triethylamine: ≧99%, Sigma-Aldrich (catalog no. T0886) orequivalent.Mobile Phase:

Add 70 mL 2-propanol and 930 mL heptane (measured separately with a 100mL and 1000-mL graduated cylinders) and 1.0 mL triethylamine (measuredwith volumetric glass pipette) to an appropriate flask and mix. Degasin-line during use.

Diluting Solution: 2-Propanol

HPLC Parameters: HPLC column: Chiralcel OD, 4.6 × 250 mm, 10 μm, DaicelChemical Industries, Ltd., catalog no. 14025. Temperature: 35° C. Flowrate*: 0.8 mL/min Gradient: NA Injection Volume: 5 μL Detection: 262 nmUV Data acquisition time: 30 min Total run time: 30 min Column MaximumPressure: 35 Bar Needle Wash: 2-propanol *Flow rate may be adjusted ±0.2ml/min to obtain specified retention times.

Retention Times for Analytes and Impurities: Retention Time CompoundPeak (Relative Retention Time, RRT)

20.6 min ± 10% (RRT 1.00)

(Enantiomer) 19.2 min (RRT 0.93)

A Typical Chromatogram from a Chiral HPLC Isolation of Compound 1-(20)is presented in FIG. 1.

1-(21) Synthesis ofN-((3S,4R,5S)-3-(2-fluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)-tetrahydrofuran-3-ylcarbamothioyl)benzamide

To chiral salt((2S,3R,4S)-4-Amino-4-(2-fluorophenyl)-2-(trifluoromethyl)tetrahydrofuran-3-yl)methanol(2S,3S)-2,3-bis(benzoyloxy)succinate (0.361 kg, 0.556 mol) was addedEtOAc (1.08 L) and the suspension was cooled to −3° C. 1.0 N aq. NaOH(1.30 L) was added over 20 mins while maintaining T<5° C. After 5 mins,benzoyl isothiocyanate (80.0 mL, 594 mmol) was added over 8 mins whilemaintaining T<5° C. After 1 h, EtOAc (722 mL) was charged. The aq. layerwas removed, and the organics were washed with sat. aq. NaHCO₃ (361 mL)and sat. aq. NaCl (361 mL). The organics were filtered over celite (90g) and rinsed with EtOAc (360 mL). The organics were concentrated undervacuum to afford a residue which was re-dissolved into CH₂Cl₂ (1.1 L)and concentrated to afford the title compound as yellow foam (261 g, 99%yield accounting for residual solvents) which was used in the followingstep. ¹H NMR (500 MHz, DMSO) δ ppm 12.04 (s, 2H), 11.20 (s, 2H), 7.95(d, J=7.4 Hz, 2H), 7.69-7.60 (m, 1H), 7.56-7.42 (m, 2H), 7.37-7.28 (m,1H), 7.24-7.12 (m, 2H). 5.59 (t, J=4.5 Hz, 1H), 5.03 (d, J=9.7 Hz, 1H),4.92 (d, J=9.7 Hz, 1H), 4.75-4.63 (m, 1H), 3.92-3.74 (m, 2H), 2.77-2.66(m, 1H); ¹³C NMR (125 MHz, DMSO) δ ppm 179.98, 167.85, 159.75 (d,J_(CF)=245.0 Hz), 133.44, 132.58, 129.88, 129.81, 129.04, 128.85, 126.31(d, J_(CF)=9.8 Hz), 124.36, 116.83 (d, J_(CF)=23.4 Hz), 76.11 (q,J_(CF)=31.0 Hz). 74.37 (d, J_(CF)=6.1 Hz), 68.77 (d, J_(CF)=3.4 Hz),57.03, 52.23.

HRMS Calculated for C₂₀H₁₈F₄N₂O₃S [M+H]⁺ 441.0896; found 441.0818.

1-(22) Synthesis ofN-((4aS,5S,7aS)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide

A solution ofN-((3S,4R,5S)-3-(2-fluorophenyl)-4-(hydroxymethyl)-5-(trifluoromethyl)-tetrahydrofuran-3-ylcarbamothioyl)benzamide(258.3 g, 583.8 mmol) in CH₂Cl₂ (1.55 L) was cooled to −19.4° C.Pyridine (118 mL, 1.46 mol) was added while maintaining temperature at−20° C., and then the reaction mixture was cooled to −24° C. In anothernitrogen purged vessel, CH₂Cl₂ (258 mL) was added followed bytrifluoromethanesulfonic anhydride (108.0 mL, 642.2 mmol). The resultingsolution was added to the reaction mixture over 30 min, whilemaintaining temperature<−19.7° C. Upon completed addition, the reactionmixture was stirred for 30 min at −20° C. to −15° C., and then warmed to−11° C. over 20 min. Saturated aq. NH₄Cl (646 mL) and water (390 mL) wasadded. The mixture was warmed to ambient temperature and the aq. layerwas removed. The organics were washed with premixed saturated aq. NH₄Cl(646 mL) and water (390 mL). The aq. layers were combined, and extractedonce with CH₂Cl₂ (520 mL). The organics were combined, and concentratedunder vacuum to afford a light orange foam (250 g, 100%). The residuewas used in the next stage without purification. ¹H NMR (500 MHz, CDCl₃)δ ppm 8.03 (d, J=6.7 Hz, 2H), 7.52 (t, J=7.0 Hz, 1H), 7.48-7.31 (m, 4H),7.20 (t, J=7.4 Hz, 1H), 7.12 (dd, J=12.0, 8.4 Hz, 1H), 4.82-4.73 (m,1H), 4.60 (d, J=8.9 Hz, 1H), 4.03 (d, J=8.3 Hz, 1H), 3.57 (d, J=2.7 Hz,1H), 3.20 (d, J=13.6 Hz, 1H), 2.81 (dd, J=13.8, 2.5 Hz, 1H); ¹³C NMR(125 MHz, CDCl₃) δ ppm 171.50, 159.57 (d, J_(cF)=247.2 Hz), 134.62,132.49, 130.65 (d, J_(CF) J=8.8 Hz), 129.77, 128.51, 128.45, 125.14 (q,J_(CF)=281.8 Hz), 124.97 (d, J_(CF)=3.0 Hz), 124.66 (d, J_(CF)=10.3 Hz),117.05 (d, J_(CF)=23.5 Hz), 66.81 (d, J_(CF)=5.2 Hz), 38.90, 23.20.

HRMS Calculated for C₂₀H₁₆F₄N₂O₂S [M+H]⁺ 425.0947; found 425.0945.

1-(23) Synthesis of(4aS,5S,7aS)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

To a solution ofN-((4aS,5S,7aS)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-yl)benzamide(250.2 g, 589.5 mmol) in methanol (1.25 L) was added K₂CO₃ (81.5 g,590.0 mmol). The suspension was heated to 65° C. for 6 h. Upon coolingto ambient temperature, the solvent was evaporated under vacuum. To theresulting residue, was added 1.0 N aq NaOH (1.18 L) and THF (502 mL).The heterogeneous mixture was heated to 45° C. for 1 h. The mixture wascooled to ambient temperature, and EtOAc (1.38 L) was added. The aqueouslayer was extracted with EtOAc (0.75 L). The organics were combined,washed with saturated aq. NaHCO₃ (500 mL) and saturated aq. NaCl (500mL). The organics were concentrated under vacuum to afford the titlecompound as a brown oil (184.1 g, 91.6% yield accounting for residualsolvents). ¹H NMR (500 MHz, DMSO) δ ppm 7.49-7.42 (m, 1H), 7.40-7.33 (m,1H), 7.26-7.15 (m, 2H), 6.26 (s, 2H), 4.77-4.54 (m, 1H), 4.40 (d, J=8.0Hz, 1H). 3.80 (dd, J=7.9, 2.3 Hz, 1H), 3.24-3.17 (m, 1H), 3.00 (dd,J=13.9, 3.2 Hz, 1H), 2.85 (dd, J=13.9, 3.9 Hz, 1H); ¹³C NMR (125 MHz,DMSO) δ ppm 159.75 (d, J_(CF)=245.1 Hz), 149.51, 131.31 (d, J_(CF)=3.9Hz), 130.13 (d, J_(CF)=8.8 Hz), 128.08 (d, J_(CF)=10.4 Hz), 128.28 (q,J_(CF)=282.1 Hz). 124.87 (d, J_(CF)=3.0 Hz), 116.80 (d, J=23.8 Hz).78.77, 76.80 (q, J_(CF)=30.8 Hz), 66.31, 36.37, 23.27.

HRMS Calculated for C₁₃H₁₂F₄N₂OS [M+H]⁺ 321.0685; found 321.0677.

1-(24) Synthesis of(4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-aminehydrochloride

To a cooled vessel containing(4aS,5S,7aS)-7a-(2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(184.1 g, 574.8 mmol) was added trifluoroacetic acid (0.954 kg) inportions while the temperature was maintained below 20° C. The mixturewas cooled to 3.5° C. and sulfuric acid (146 mL, 2.73 mol) was addedover 20 min while the temperature was maintained below 5° C. Fumingnitric acid (39.8 mL, 0.948 mol) was added over 30 min, while thetemperature was maintained below 10° C. After 1.5 h at 0-10° C., thereaction mixture was slowly quenched by transferring into an aq.solution of NaOH (575 g, 14.4 mol) in water (4.6 L) cooled to 5° C. Theresulting suspension was stirred for 1 h at 21° C. The suspension wasthen filtered and the solid rinsed with cold water (920 mL). The solidwas dried under vacuum until constant weight, and then dissolved intoethanol (1.05 L). The solution was heated to 35° C., and conc. HCl (55.6mL, 0.690 mol) was added while maintaining temperature below 40° C. Thesuspension was then cooled to −5° C., held for 1 hr and filtered. Thesolid was rinsed with cold ethanol (420 mL) and dried until constantweight to obtain the title compound (185.0 g, 87.3%). ¹H NMR (500 MHz,DMSO) δ ppm 11.80 (s, 2H), 8.45-8.36 (m, 1H), 8.31 (dd, J=6.6, 2.5 Hz,1H), 7.66 (dd, J=11.1, 9.3Hz, 1H), 4.96-4.72 (m, 1H), 4.58 (d, J=10.0Hz, 1H), 4.27 (d, J=9.9 Hz, 1H), 3.76-3.66 (m, 1H), 3.39 (dd, J=14.9,3.6 Hz, 1H), 3.24 (dd, J=14.3, 4.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO) δppm 168.34, 163.33 (d, J_(CF)=257.8 Hz), 144.58, 127.61 (d, J_(CF)=11.6Hz), 125.84, 124.10, 119.28 (d, J_(CF)=26.5 Hz), 77.38 (q, J_(CF)=31.5Hz), 75.99, 65.88 (d, J_(CF)=4.8 Hz), 40.36, 23.98.

HRMS Calculated for C₁₃H₁₁F₄N₃O₃S [M+H]⁺ 366.0536; found 366.0523.

1-(25) Synthesis of(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine

Ethanol (0.975 L) was added to iron powder (62.5 g, 1.12 mol) undernitrogen atmosphere. Concentrated HCl (9.03 mL) was added at ambienttemperature and the suspension was heated to 65° C. for 1.5 h. Thesuspension was then cooled to 50° C., and sat. aq. NH₄Cl (299 g) wereadded. The temperature of the reaction mixture was allowed to reach 50°C., and(4aS,5S,7aS)-7a-(2-fluoro-5-nitrophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-aminehydrochloride (75.0 g, 187.0 mol) was added in portions whilemaintaining temperature below 68° C. After 30 min, ethanol (0.45 L) wasadded, and the reaction mixture was cooled to 20-25° C. over 1 h. Thesuspension was stirred for 2 h and filtered over Celite (75 g) rinsingwith ethanol (0.972 L). The solution was concentrated under vacuum to abrown solid. Water (0.9 L) was added followed by 3.0 N NaOH (0.187 L,560 mmol) while maintaining temperature below 35° C. The resultingsuspension was stirred for 1 h at 20-25° C. The suspension was filtered,and the solid was rinsed with cold water (0.38 L). The solid was driedunder vacuum at 40-45° C. over 24 h to obtain the title compound (57.7g, 95.5%). ¹H NMR (500 MHz, DMSO) δ ppm 6.81 (dd, J=12.5, 8.6 Hz, 1H),6.62 (dd, J=7.0, 2.9 Hz, 1H), 6.50-6.42 (m, 1H), 6.16 (s, 2H), 4.96 (s,2H), 4.72-4.54 (m, 1H), 4.35 (d, J=7.8 Hz, 1H), 3.74 (dd, J=7.8, 2.5 Hz,1H), 3.18-3.08 (m, 1H), 3.01 (dd, J=13.9, 3.0 Hz, 1H). 2.84 (dd, J=13.8,3.8 Hz, 1H); ¹³C NMR (125 MHz, DMSO) δ ppm 156.20 (d, J_(CF)=243.0 Hz),148.73, 145.49, 127.86 (d, J_(CF)=11.0 Hz), 116.79 (d, J_(CF)=24.8 Hz),116.10 (d, T_(CF)=3.3 Hz), 114.10 (d, J_(CF)=8.0 Hz), 78.89, 76.57 (q,J_(CF)=31.0 Hz), 66.35, 36.35, 23.11.

HRMS Calculated for C₁₃H₁₃F₄N₃OS [M+H]⁺ 336.0794; found 336.0789.

The title compound was subjected to an Ames test (Salmonella typhimuriumtester strains TA98, TA100, TA1535 and TA1537 and Escherichia colitester strain WP2 uv. Mutation Research 1975, 31, 347; Mutation Research1976, 38, 3; Proc. Nat. Acad. Sci. USA 1976, 73, 950; Proc. Nat. Acad.Sci. USA 1975, 72, 5135) in the absence and presence of rat liver S9.The compound was negative up to the highest dose/concentration tested(5000 ug/plate).

1-(26) Synthesis ofN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide

A suspension of 5-methoxypyrazine-2-carboxylic acid (26.29 g, 0.17 mol)in N,N′-dimethylimidazoline-2-one (160 mL) was stirred at ambienttemperature for 15 min, then cooled to 2.2° C. Thionyl chloride (14.7mL, 0.202 mol) was added while maintaining temperature under 5° C. Theresulting suspension was stirred at 0-10° C. for 2 h while ittransitioned to a clear solution. In another vessel,(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(52.0 g, 0.155 mol) was dissolved into N,N′-dimethylimidazoline-2-one(160 mL). The resulting solution was added to the solution of acylchloride while maintaining temperature below 10° C. The reaction mixturewas stirred for 30 min. Water (780 mL) was charged while maintainingtemperature below 30° C. The resultion mixture was stirred for 30 min,and then EtOAc (780 mL) was added. To this mixture was added, 50% aq.NaOH (84.8 g) until the pH of the aqueous layer reached 11. The aq.layer was extracted with EtOAc (260 mL). The organics were combined,washed with sat. aq. NaCl (260 mL) and water (260 mL). The organics werefiltered over Celite pad (26 g) and rinsed with EtOAc (260 mL). Theorganics were concentrated under vacuum to afford a solid. To the solidwas added 1-propanol (728 mL), and the suspension was heated to 75° C.until a clear solution formed. The solution was cooled to −10° C. andheld for 1 h. The solid was filtered, rinsed with cold 1-propanol (104mL) and dried under vacuum (35° C.) until constant weight to afford thetitle compound (62.1 g, 84.9%). ¹H NMR (500 MHz, DMSO) δ ppm 10.56 (s,2H), 8.88 (d, J=1.2 Hz, 1H), 8.39 (d, J=1.2 Hz, 1H), 7.95-7.83 (m, 2H),7.18 (dd, J=12.0, 8.8 Hz, 1H), 6.25 (s, 2H), 4.76-4.60 (m, 1H), 4.36 (d,J=8.1 Hz, 1H), 4.01 (s, 3H), 3.88 (dd, J=7.9, 2.3 Hz, 1H), 3.23-3.11 (m,2H), 2.91 (dd, J=13.8, 3.6 Hz, 1H); ¹³C NMR (125 MHz, DMSO) δ ppm162.11, 161.93, 156.13 (d, J_(CF)=242.9 Hz), 149.38, 142.01, 138.35,135.09, 133.98, 128.53 (d, J_(CF)=11.6 Hz), 126.06 (q, J_(CF)=282.0 Hz),123.32, 121.93 (d, J_(CF)=8.6 Hz), 116.76 (d, J_(CF)=25.1 Hz), 78.86 (d,J_(CF)=6.9 Hz), 76.94 (q, J_(CF)=30.5 Hz), 66.37, 54.75, 36.44, 23.53.

HRMS Calculated for C₁₉H₁₇F₄N₅O₃S [M+H]⁺ 472.1066; found 472.1052.

Specific optical rotation [α]_(D)+110.5 (c 0.519, MeOH)

Specific Optical Rotation Parameters:

Equipment: Polarimeter: Perkin Elmer, model 341 or equivalent. Cell:Microglass cell, 100 mm pathlength, 1.0 mL capacity, Perkin-Elmer Cat.#B001-7047. Balance: Calibrated analytical balance capable of weighing±0.1 mg Water Bath: NESLAB RTE 1121 Chiller or equivalent. VolumetricClass A. glassware: Quartz Standard ID number 098799, or equivalent.Polarimeter: Perkin Elmer, model 341 or equivalent. Reagents: Methanol:HPLC grade, Baker (catalog no. 9093-03) or eqivalent Instrumentparameters: Lamp: Na/Hal, Perkin-Elmer Cat. #B000-8754. Cell: Microcell(100 mm), Perkin-Elmer Cat. #B004-1693. Cell Path: 100 mm (1 decimeter)Mode: OROT Wavelength: 589 nm Cell Temperature: 20° C. Integration time:2 seconds Aperture: MICRO Water bath 20 ± 1° C. temperature:

Alternative Preparation ofN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamide(Example 8)

5-(Fluoromethyl)pyrazine-2-carboxylic acid (32.6 g, 1.05 equiv) and(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amine(70.0 g, 1.0 equiv)¹ were charged to a reactor and ethyl acetate (EtOAc,630 mL) was added to the mixture to give a suspension. A solution ofn-propane phosphonic acid anhydride (T3P, 146 g, 1.10 equiv, 50 wt % inEtOAc) was added at ambient temperature while controlling the internaltemperature below 30° C. The reaction mixture was stirred at 40-45° C.>3hours and monitored by HPLC. The reaction mixture was cooled to 15-20°C. and water (140 mL) was charged. After 10-15 minutes charged 28%ammonium hydroxide (175 mL) while controlling the temperature below 30°C. EtOAc (245 mL0 was added and the reaction mixture was stirred for 30minutes at ambient temperature. The aqueous phase was separated andback-extracted with EtOAc (490 mL). The organic phases were combined andwashed with 15% aq. NaCl (140 mL) and water (140 mL). The organic layerwas filtered over Celite (1.0 Wt) and rinsed with EtOAc (140 mL). Thesolution was concentrated under vacuum to obtain a beige solid(quantitative crude yield) which was recrystallized from 1-propanol toaffordN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-(fluoromethyl)pyrazine-2-carboxamideas a white solid (70.0 g). ¹H NMR (500 MHz, DMSO) δ 10.89 (s, 1H), 9.30(s, 1H), 8.89 (s, 1H), 7.95 (dd, J=7.3, 2.7 Hz, 1H), 7.94-7.89 (m, 1H),7.21 (dd, J=12.0, 8.8 Hz, 1H), 6.22 (s, 2H), 5.71 (d, J=46.3 Hz, 2H),4.77-4.61 (m, 1H), 4.37 (d, J=8.1 Hz, 1H), 3.87 (dd, J=8.0, 2.7 Hz, 1H),3.20 (dt, J=7.0, 3.5 Hz, 1H), 3.15 (dd, J=13.9, 3.1 Hz, 1H), 2.91 (dd,J=13.8, 3.8 Hz, 1H). ¹³C NMR (126 MHz, DMSO) δ161.32 (s), 155.82 (d,J=243.4 Hz), 153.71 (d, J=18.7 Hz), 148.77 (s), 144.71 (d, J=1.9 Hz),143.30 (s), 141.01 (d, J=5.6 Hz), 134.36 (d, J=2.0 Hz), 128.20 (d,J=12.1 Hz), 125.57 (q, J=283.0 Hz), 123.12 (d, J=3.6 Hz), 121.64 (d,J=8.6 Hz), 116.35 (d, J=25.2 Hz), 82.55 (d, J=165.8 Hz), 78.37 (s),76.44 (q, J=30.6 Hz), 65.89 (d, J=5.3 Hz), 35.89 (s), 23.01 (s).

HRMS Calculated for C₁₉H₁₇F₅N₅O₂S [M+H]⁺ 474.1023; found 474.1032.

Specific Optical Rotation: [α]_(D) ²⁰=+102.4

¹A preparation of(4aS,5S,7aS)-7a-(5-amino-2-fluorophenyl)-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-2-amineis described herein above in step 1-(25) in the alternative preparationofN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide(Example 1).

In Vitro Cellular Assay:

Quantification of Aβ Peptide in Culture of Neurons from Rat Fetus Brain

(1) Rat Primary Neuronal Culture

Primary neuronal cultures were prepared from the cerebral cortex ofembryonic day 18 Wistar rats (Charles River, UK). Specifically, theembryos were aseptically removed from pregnant rats under etheranesthesia. The brain was isolated from the embryo and immersed in HBSS(Sigma Aldrich #H9269) containing 10 mM HEPES (Gibco #15630-056). Thecerebral cortex was collected from the isolated brain under astereoscopic microscope. The cerebral cortex fragments collected wereenzymatically treated in an enzyme solution containing 0.05%trypsin-EDTA solution (GIBCO, #25300) at 37° C. for 20 minutes todisperse the cells. The cells were then washed twice and then gentlyresuspended in Neurobasal medium (Gibco #21103) supplemented with 2% B27supplement (GIBCO #17504-044), 0.5 mM L-glutamine (GIBCO #25030), 1×N2(GIBCO #17502-048), 100 ug/ml Pen/Strep (GIBCO 15140-122) and 5% heatinactivated FCS (PAA #A15-701). The cell dispersion was filtered througha 40-μm nylon mesh (BD Falcon #352340) to remove the remaining cellmass, and thus a neuronal cell suspension was obtained. The neuronalcell suspension was diluted with the medium above and then plated in avolume of 100 μL/well at an initial cell density of 3.25×10⁵ cells/ml inpoly-D-lysine coated 96-well culture plate (Greiner #655940). The platedcells were cultured in the culture plate at 37° C. in 5% CO₂-95% air for24 hrs. The total amount of the medium was replaced with ‘assayNeurobasal medium’ (as above excluding heat inactivated FCS), and thenthe cells were cultured for a further five days.

(2) Addition of Compound

The drug was added to the culture plate on Day 6 of culture as follows.8 point compound serial dilutions were generated in DMSO at aconcentration of ×1000 that of the final assay concentration (FAC).Compound solutions were then prepared by adding 999 ul of ‘AssayNeurobasal media’ (as described in above section) to 1 ul of DMSOcompound stock. The total amount of the medium was removed from each ofthe cell plate wells, and 140 μL/well of ‘Assay Neurobasal media’ wasadded followed by 60 ul of compound solution. The final DMSOconcentration was 0.1%.

(3) Sampling

The cells were cultured for either 1 or 3 days after addition of thecompound for ABx-40 and ABx-42 assays respectively. 150 μl of samplemedium was collected and used as the ELISA sample.

(4) Evaluation of Cell Survival

Cell survival was evaluated using an Alamar assay according to thefollowing procedure. After collecting the sample to be used in the ELISAassay, 50 μl of 20% Alamar blue solution (Invitrogen #DAL1100) in assayNeurobasal media, was added to 50 μl of remaining sample within eachwell. Cells were then incubated at 37° C. in 5% CO₂-95% air for 1 hr.

Measurement of fluorescence intensity for each well was the carried outat 540/590 nm using a Pherastar plus plate reader (BMG labtech). Uponmeasurement, wells having no cells plated and containing only the mediumand Alamar solution were set as background (bkg).

(5) Aβ ELISA

Human/Rat β Amyloid (42) ELISA Kit Wako (#290-62601) and Human/Rat βAmyloid (40) ELISA Kit Wako (#294-62501) from Wako Pure ChemicalIndustries, Ltd. were used for Aβ ELISA. Aβ ELISA was carried outaccording to the protocols recommended by the manufacturers, describedin the documents accompanying the kits. The results were shown aspercentage of the control groups and IC₅₀ values for each compound weredetermined using four parameter logistic fit model using the XLFITSsoftware package (IDBS).

The compounds of the present invention have an Aβ42 production reducingeffect.

The compound of the general formula (I) or pharmaceutically acceptablesalt thereof according to the present invention has an Aβ42 productionreducing effect. Thus, the present invention can particularly provide aprophylactic or therapeutic agent for a neurodegenerative disease causedby Aβ such as Alzheimer-type dementia or Down's syndrome.

As measured by the above in vitro assay, compound Examples 1 to 18showed IC₅₀ values of less than 0.1 μM as shown in Table 5:

TABLE 5 Example IC₅₀ (uM) 1 0.008 2 0.004 3 0.004 4 0.008 5 0.012 60.006 7 0.008 8 0.006 9 0.007 10 0.010 11 0.010 12 0.006 13 0.044 140.002 15 0.007 16 0.009 17 0.051 18 0.015

The invention claimed is:
 1. A compound which isN-(3-((4aS,5S,7aS)-2-amino-5-(trifluoromethyl)-4a,5,7,7a-tetrahydro-4H-furo[3,4-d][1,3]thiazin-7a-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound according to claim 1, or apharmaceutically acceptable salt thereof, as an active ingredient inassociation with a pharmaceutically acceptable carrier.
 3. A method oftreating Down's syndrome, comprising administering to a human subjectsuffering from Down's syndrome an effective amount of the compoundaccording to claim 1, or a pharmaceutically acceptable salt thereof. 4.A method of treating Alzheimer-type dementia, comprising administeringto a human subject suffering from Alzheimer-type dementia an effectiveamount of the compound according to claim 1, or a pharmaceuticallyacceptable salt thereof.